Excessive-sucrose diets contribute to brain angiopathy and better brain dysfunctions
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Shinobu Hirai https://orcid.org/0000-0002-2354-3759 [email protected], Hideki Miwa https://orcid.org/0000-0001-6015-3007, Tomoko Tanaka https://orcid.org/0000-0003-2073-1487, Kazuya Toriumi https://orcid.org/0000-0002-8593-3269, Yasuto Kunii https://orcid.org/0000-0003-1569-7819, Hiroko Shimbo https://orcid.org/0000-0003-1677-752X, Takuya Sakamoto, Mizuki Hino https://orcid.org/0000-0003-3700-0322, Ryuta Izumi https://orcid.org/0000-0002-8453-3474, Atsuko Nagaoka https://orcid.org/0000-0002-1159-4436, Hirooki Yabe https://orcid.org/0000-0003-2668-129X, Tomoya Nakamachi https://orcid.org/0000-0003-3212-3198, Seiji Shioda, Takashi Dan, Toshio Miyata https://orcid.org/0000-0001-9153-5024, Yasumasa Nishito https://orcid.org/0000-0001-7621-6172, Kazuhiro Suzuki https://orcid.org/0000-0002-0038-4329, Mitsuhiro Miyashita, Toshifumi Tomoda, Takatoshi Hikida, Junjiro Horiuchi, Masanari Itokawa https://orcid.org/0000-0003-4433-8030, Makoto Arai https://orcid.org/0000-0003-3400-9815, and Haruo Okado https://orcid.org/0000-0001-5674-420X [email protected]

Summary

Metabolic dysfunction is idea to contribute to the severity of psychiatric concerns; nonetheless, it has been unclear whether latest high–straightforward sugar diets contribute to pathogenesis of those illnesses. Here, we masks that a high-sucrose diet in the course of formative years induces psychosis-associated behavioral endophenotypes, including hyperactivity, miserable working memory, impaired sensory gating, and disrupted interneuron feature in mice miserable for glyoxalase-1 (GLO1), an enzyme concerned about detoxification of sucrose metabolites. Furthermore, the high-sucrose diet prompted microcapillary impairments and reduced brain glucose uptake in brains of Glo1-miserable mice. Aspirin estimable in difference angiopathy, making improvements to brain glucose uptake and combating irregular behavioral phenotypes. Identical vascular pain to our mannequin mice used to be found in the brains of randomly mute schizophrenia and bipolar disorder sufferers, suggesting that psychiatric concerns are associated with angiopathy in the brain precipitated by diversified environmental stresses, including metabolic stress.

INTRODUCTION

Smitten by the realm lift in consumption of straightforward sugars, the World Health Organization printed guidelines that addressed concerns referring to body weight in discovering and dental caries trend in 1995 (1). Excessive sugar intake by myself will enhance the risks of a gigantic sequence of chronic illnesses, including diabetes, hypertension, and kidney illness. On the other hand, there are few analysis on the effects of high sugar intake in the course of formative years on future psychological health. The each day caloric intake from straightforward sugar by teenagers is better than that seen for assorted age groups (~20% of total each day caloric intake) (2). Most chronic psychiatric concerns, including schizophrenia (SZ) and bipolar disorder (BD) discussed in the latest in discovering, create before the age of 30 thru complex interactions among more than one genetic and environmental threat components. Sufferers with SZ and BD tell approximately twofold more sugar than age-matched wholesome folk, and sufferers with SZ who tell more sucrose masks more severe signs (35). Furthermore, the odds ratios for psychological damage, hyperactivity, and behavioral concerns were one of the best among teens who self-reported one of the best consumption of nonetheless drinks (6).

Estimable glycation cease products (AGEs) and reactive carbonyl compounds are produced from irreversible nonenzymatic tainted-linking reactions between carbonyl-containing molecules (e.g., sugars and reactive carbonyl compounds) and numerous molecules (e.g., amino groups of proteins), and AGEs can lead to the induction of inflammation and oxidative stress in diversified tissues, main to a obvious solutions loop that additional will enhance AGEs (7). There might be big evidence that sufferers with psychiatric concerns enjoy elevated proinflammatory cytokines [e.g., interleukin-1β (IL-1β) and IL-6] and increased oxidative stress (811). Glyoxalase I (GLO1) is a zinc metalloenzyme that protects cells from AGE toxicity by catalyzing the response between the reactive carbonyl compound methylglyoxal to glutathione to develop S-lactoyl-glutathione (12). Sufferers with depressive-bid BD and main depressive disorder enjoy reduced quantities of GLO1 mRNA, suggesting that this reduction, in combination with a rise in sugar consumption, might be a explanation for the increased inflammation and oxidative stress seen in these illnesses (13). Furthermore, a affected person with SZ exhibiting miserable convalescence is found to harbor a frameshift mutation in GLO1 main to reduced enzyme process (1416).

Regardless of gathering evidence, it’s nonetheless unproven that excessive sugar intake contributes to the pathogenesis of psychiatric concerns among inclined folk. We addressed this request by producing mice with psychosis-associated phenotypes associated with the gene × ambiance interactions (G × E) to resolve whether excessive sucrose consumption in the course of formative years is a recent environmental threat yell for SZ and BD. We additional identified angiopathy in mice with high sucrose intake and genetic vulnerability and confirmed identical vascular pain in randomly mute brain samples from sufferers with BD and SZ. This implies that a gigantic selection of genetic and environmental components can adversely enjoy an affect on brain capillaries. As smartly as, we found that chronic anti-inflammatory drug remedy prevented core phenotypes of psychiatric concerns and vascular pain, suggesting that angiopathy can even unprejudiced enjoy a extraordinarily significant affect on the plot and/or situation of psychiatric concerns.

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RESULTS

Psychosis-associated behavioral phenotypes in mice on a high-sucrose diet

We fed mice one in all two diets containing the identical total energy and caloric proportions of carbohydrates, plump, and proteins but with either starch or sucrose as the main carbohydrate (Fig. 1A). We investigated four groups of mice fed these diets for 50 days straight after weaning (from postnatal day 21, equivalent to the juvenile/adolescent stage): starch-fed wild-form (WT) mice [control (CTL)], sucrose-fed WT mice [environmental stressor (Env)], starch-fed Glo1 heterozygous knockout (Glo1/+) mice [genetic factor (Gen)], and sucrose-fed Glo1/+ mice (G × E) (Fig. 1A). Diminished GLO1 expression in Glo1/+ mice used to be confirmed in the cerebral cortices, including the hippocampus, using Western blotting (fig. S1, A and B). The body weight trajectories of those mice were identical to those of preserve an eye on mice for no longer decrease than 11 weeks of age, indicative of regular structural trend (fig. S1C) and inserting off obesity as a seemingly explanation for the seen team differences described beneath. We tested eight assorted behaviors in mice, including starting up-field (OF) locomotor process (Fig. 1B), acoustic startle response (Fig. 1C), prepulse inhibition (PPI; a measure of sensory-motor gating) (Fig. 1D), object space performance (frail as a take a look at of working memory) (Fig. 1E), self-grooming (Fig. 1F), nest constructing (Fig. 1G), elevated plus maze process (fig. S2A), and social interaction (fig. S2B). These behaviors might be categorised into folk who’re no longer suffering from either diet or genotype, folk who’re suffering from genotype but no longer diet, folk who’re suffering from diet but no longer genotype, and folk that are suffering from the mix of diet and genotype. Thus, neither diet, genotype, nor a mix of diet and genotype had any significant create on social interactions (fig. S2B), whereas acoustic startle responses were reduced in Glo1/+ mice but no longer vastly suffering from feeding form (Fig. 1C). Self-grooming (Fig. 1F), nest constructing (Fig. 1G), and elevated plus maze process (fig. S2A) were suffering from diet, but no longer genotype, whereas OF locomotor process, PPI, and object space performance required a mix of sucrose feeding and a Glo1/+ genotype to be affected. Within the OF take a look at (Fig. 1B), neither starch-fed Glo1/+ mice nor sucrose-fed WT mice confirmed significant differences in distance traveled in contrast with starch-fed WT mice, whereas sucrose-fed Glo1/+ mice confirmed significant will enhance. Equally, starch-fed Glo1/+ mice and sucrose-fed WT mice had identical PPI ratings in contrast with starch-fed WT mice, whereas sucrose-fed Glo1/+ mice confirmed significant decreases (Fig. 1D). Sucrose-fed WT mice had a nonsignificant reduction in object space take a look at (OLT) ratings, and this reduction became significant in sucrose-fed Glo1/+ mice (Fig. 1E). Together, our outcomes imply that a mix of sucrose feeding and Glo1/+ mutation can generate recent phenotypes paying homage to psychiatric illness phenotypes that are no longer found by altering diet or genotype by myself (table S1).

Fig. 1. Technology of G × E mannequin mice and psychosis-associated phenotype analyses.

(A) Experimental timeline. Glimpse Supplies and Recommendations for a complete description. (B to G) Behavioral analyses in the four groups of mice (n = 18 to 23 mice per team). (B) Spontaneous locomotor process in the OF take a look at. (C) Acoustic startle responses. (D) PPI using a 70-dB prepulse. (E) OLT to purchase into consideration working memory. (F) Length of self-grooming in the home cage. (G) Quantification of nest-constructing abilities over 8 hours. Post hoc Dunnett’s more than one comparisons take a look at among groups at on every occasion point signifies ###P < 0.001 for Suc Glo1/+ versus Ctrl (Starch +/+) and $$P < 0.01 for Suc +/+ versus Ctrl (Starch +/+). (H and J) Extracellular dopamine (DA) focus in the NAc measured at 20-min intervals using an in vivo microdialysis system in the presence or absence of aripiprazole (Aripi). Methamphetamine (1.25 mg/kg) used to be administered thru intraperitoneal injection at time 0 (arrow) (n = 6 to 11 mice per team). Glimpse Supplies and Recommendations for detailed statistical analyses. (I) Outcomes of Aripi remedy on locomotor process (n = 12 to 18 mice per team). (B to H) Starch +/+ team used to be frail as a preserve an eye on for put up hoc Dunnett’s take a look at. All recordsdata are presented as components ± SEM. P < 0.001, P < 0.01, and *P < 0.05.

To additional toughen our view that synergistic G × E interactions can even unprejudiced induce or enhance the severity of psychosis-take care of signs, we examined the effects of the high-sucrose diet on an additional main psychosis-associated mutation in Disrupted-in-schizophrenia 1 (DISC1) locus (fig. S3) (17). DISC1 performs significant roles in neurodevelopment and synaptic process, but a loss-of-feature mutation in DISC1 gene by myself is no longer enough to cause psychiatric concerns (18). A lot just like the outcomes for Glo1/+ mice, Disc1/+ mice fed with a high-sucrose diet (G × E mice) confirmed severely impaired object space performance (working memory) and PPI (fig. S3, C and D), whereas WT mice fed with a high-sucrose diet or Disc1/+ mice fed with preserve an eye on diet didn’t, as soon as more suggesting that a mix of genetic and environmental influences can induce psychosis-take care of behaviors.

SZ is expounded with increased amphetamine-prompted dopamine starting up in the striatum (19, 20) and unbalanced dopamine regulation between the medial prefrontal cortex (mPFC) and striatum (21). To see the effects of high-sucrose diet on dopamine starting up in Glo1/+ mice, we subsequent measured dopamine ranges in the nucleus accumbens (NAc) and mPFC of those mice using in vivo microdialysis. We found that the high-sucrose diet increased each basal and amphetamine-prompted dopamine starting up in the NAc specifically in Glo1/+ mice (Fig. 1H) whereas reducing basal dopamine starting up in the mPFC in either genetic background (fig. S2C). To assess whether enhanced dopamine starting up in the NAc can even induce the seen behavioral phenotypes, we examined the effects of aripiprazole, a D2 receptor partial agonist and medical antipsychotic (22), administered in the course of the final 7 days (0.5 mg/kg per day) of sucrose feeding (Fig. 1, A, I, and J, and fig. S2, D to I). We found that the hyperlocomotion and increased striatal dopamine starting up in G × E mice were fully reversed by aripiprazole remedy (Fig. 1, I and J), whereas assorted behavioral defects were no longer improved (fig. S2, D to I). Thus, aripiprazole remedy selectively improves a subset of behavioral abnormalities in G × E mice that are doubtless precipitated by dysregulated dopamine signaling.

Dysfunction of parvalbumin-obvious inhibitory interneurons in G × E mice

A reduction in density of parvalbumin (PV)–obvious GABAergic interneurons is reported for SZ (2326), and the precisely coordinated process of those neurons is vital for the regulation of PPI and working memory (27). Thus, we subsequent investigated PV expression using immunohistochemistry and Western blotting to resolve whether alterations in PV signaling can even unprejudiced contribute to our seen psychiatric illness–associated phenotypes. A high-sucrose diet precipitated a discount in PV-obvious cells in each Glo1/+ and WT mice (Fig. 2, A and B) and vastly reduced PV expression in sucrose-fed Glo1/+ mice (Fig. 2, C and D), suggesting that the sucrose diet and the Glo1/+ mutation had additive or synergistic effects on PV signaling. Since gamma oscillations are produced thru the synchronous activation of PV neurons, we measured gamma oscillations (30 to 45 Hz) using surface electroencephalography (EEG) to resolve whether down-regulation of PV protein used to be accompanied by purposeful abnormalities in neural process. Sucrose-fed mice (WT and Glo1/+ mice) exhibited elevated baseline gamma oscillation energy in contrast with preserve an eye on mice (starch-fed WT) in the home cage (Fig. 2E). Furthermore, most keen sucrose-fed Glo1/+ mice failed to masks a rise in the gamma oscillation energy when drawing shut a recent object (Fig. 2F). These outcomes are in maintaining with findings in sufferers with SZ and BD as smartly as assorted mouse models of psychosis demonstrating increased baseline gamma oscillations and diminished sensory stimulus–evoked synchronized gamma energy (28, 29). Thus, our outcomes imply that G × E mice mimic the pathophysiological changes of PV neurons seen in psychiatric concerns.

Fig. 2. PV-obvious interneuron dysfunction in G × E mice.

(A) PV immunohistochemistry in the mPFC. (B) Preference of PV-obvious cells in the mPFC (n = 4 mice per team). (C) Western blot analysis of PV protein expression using tubulin as interior preserve an eye on. (D) Densitometric analysis of PV protein expression: PV band intensities were divided by the corresponding tubulin band intensities (n = 3 mice per team). (E) Moderate gamma band energy in the home cage and in the course of recent object recognition in the OF (n = 7 to 8 mice per team). (F) Adjustments in gamma band energy from the dwelling cage to the unconventional object phase in particular individual mice (n = 7 to 8 mice per team). Every P fee signifies the final consequence of repeated measures ANOVA. The create of changes in gamma energy at Starch +/+ (F1, 5 = 8.29, P = 0.035), at Starch Glo1/+ (F1, 6 = 29.75, P = 0.0016), at Sucrose +/+ (F1, 6 = 12.04, P = 0.013), and at Sucrose Glo1/+ (F1, 7 = 4.038, P = 0.084). (B, D, and H) Starch +/+ team used to be frail as a preserve an eye on for put up hoc Dunnett’s take a look at. All recordsdata are presented as components ± SEM. P < 0.001, P < 0.01, and *P < 0.05. n.s., no longer significant.

To summarize, the administration of a high-sucrose diet to Glo1/+ mice induces behavioral, histological, and pathophysiological phenotypes paying homage to phenotypes seen in psychiatric concerns. This implies that excessive sucrose intake in the course of formative years is a doable environmental threat yell for these illnesses.

AGE accumulation and impaired astrocyte feature in G × E mice

How does a decrease in GLO1 expression and a rise in sucrose consumption lead to defects in process of PV and dopaminergic neurons? We first examined GLO1 expression and detected high expression in astrocytes (fig. S4, A to G), especially those surrounding capillaries (fig. S4, B and C), life like expression in neurons (fig. S4, H and I), and low expression (beneath detection limits) in vascular endothelial cells (fig. S4, A to G) and microglial cells (fig. S4, H and J). Expression used to be no longer detected in the brains of Glo1 homozygous mice (fig. S4K), indicating that the antibody we frail used to be order for GLO1.

Since each GLO1 and a high-sucrose diet are associated with glycation toxicity, we subsequent examined AGE immunoreactivity to detect ranges of AGE products of glucose metabolism and/or chemical response of glucose. We found stronger AGE immunoreactive alerts in the vascular endothelial cells of G × E mice in contrast with preserve an eye on mice (Fig. 3, A to H). Furthermore, we detected AGE products of methylglyoxal metabolism and/or chemical response of methylglyoxal using AGE-4 antibody in the microglia of sucrose-fed mice (fig. S5, A to E). Compared to controls, AGE accumulation in sucrose-fed Glo1/+ mice used to be accompanied by elevated IBA1 immunofluorescence depth and an enlarged CD68-obvious space (fig. S5, F to H), each of which are phenotypes identified in activated microglia (30, 31). Thus, sucrose feeding in Glo1/+ mice ends up in microglial and endothelial AGE accumulation and microglial activation.

Fig. 3.

AGE accumulation in the neurovascular endothelium and pre-inflammatory set of astrocytes in G × E mice.

(A) AGE immunohistochemistry in the mPFC. (B) Size of AGE immunoreactive space. The mean depth of the AGE-immunopositive space in the complete image used to be measured for each section (n = 3 mice per team). (C to E) Colocalization of the endothelial cell marker tomato lectin with the astrocytic marker ALDH1L1 or AGEs. (F) Enlarged version of photos in (C) presenting the place frail for colocalization analysis. (G) Two-dimensional depth histogram of the two indicated channels for figuring out the colocalization of AGE with ALDH1L1 (astrocyte) or tomato lectin (endothelial cell). (H) Moderate R fee of colocalization recordsdata including (G) three assorted areas of three mice. (I) Immunohistological photos of green fluorescent protein (GFP)–obvious astrocytes and ALDH1L1 in the mPFC place and merged GFP/ALDH1L1 image. (J) Share of GFP-obvious cells per total ALDH1L1-obvious cells in each image in (I). (K) Imply fluorescent GFP intensities of 10 randomly selected cells per image in (I) (from four self sustaining mice). (B, J, and K) The Starch +/+ team used to be frail as a preserve an eye on for put up hoc Dunnett’s take a look at. All recordsdata are presented as components ± SEM. P < 0.001, P < 0.01, and *P < 0.05.

Since astrocytes express GLO1 most strongly (fig. S4, A to G), we next used mice expressing green fluorescent protein (GFP) under the control of the glial fibrillary acid protein (GFAP) promoter (32, 33) to evaluate how astrocytes are affected in our G × E condition. Typically, cellular damage from AGEs is caused by inflammatory responses induced by receptor of AGEs activation or by a loss of normal protein function following AGE-forming reactions (34). Astrocytes exhibit a well-described reactive phenotype in response to pathogenic conditions including neuroinflammation characterized by enhanced GFAP expression (35, 36). Strongly enhanced GFAP promoter function was observed in G × E mice, without changes in the number of GFAP-positive astrocytes (Fig. 3, I to K), indicating that the astrocytes in G × E mice are in the reactive precondition during the high-sucrose feeding. Together, these results suggest that a high-sucrose diet enhances AGE production in endothelial cells and microglia (Fig. 3, A to H, and fig. S5, A to E) and converts astrocytes into a pre-inflammatory state in Glo1-deficient mice (Fig. 3, I to K).

Microcapillary angiopathy and impaired glucose intake in G × E mice

Endothelial cells and astrocytes are functional and physical components of the blood-brain barrier (BBB), which tightly controls the parenchymal environment by modulating the selective passage of nutrients and various factors (37). Thus, endothelial AGE accumulation and astrocyte reactivity may impair BBB function. To examine changes in endothelial function, we first conducted a transcriptome analysis of the PFC, a region strongly implicated in psychiatric impairments, using microarrays (Fig. 4, A and B). The coagulation factor V, essential for the production of fibrin from fibrinogen, ranked seventh on a list of transcripts exhibiting more than doubled expression in G × E mice compared with the other three groups (Fig. 4A and tables S2 and S3). Fibrin controls hemostasis via polymerization with platelets to form blood clots, and deposits of this protein are indicative of endothelial abnormalities (38). In early stages of endothelial cell impairment, fibrin accumulates in capillaries. Therefore, we investigated vascular fibrin accumulation using immunohistochemistry and confirmed the presence of significant fibrin accumulation on the vascular lumen side of endothelial cells in brain capillaries of G × E mice (Fig. 4, C to G). Fibrin leakage and deposition in the brain parenchyma, as observed in Alzheimer’s disease, were not detected in G × E mice, indicating that physical BBB disruption did not occur (39).

Fig. 4. Angiopathy and impaired glucose transport in G × E mice.

(A and B) Venn graph showing overlap in PFC genes exhibiting a >2-fold (A) or <0.5-fold (B) expression switch in contrast with the preserve an eye on (CTL) team (n = 3 mice per team). (C) Immunohistochemical photos of fibrin and the endothelial cell marker tomato lectin. (D) Size of the gap of fibrin immunoexpression in (C). The mean depth of the fibrin-immunopositive space of the complete image used to be measured for each section (n = 4 mice per team). (E) Magnified immunohistochemical photos of fibrin and the endothelial cell marker tomato lectin. (F) Two-dimensional depth histogram of the two indicated channels using the left and center panels in (E) for figuring out fibrin colocalization with tomato lectin. (G) Moderate R fee of colocalization recordsdata in (F) from three assorted areas of three mice. (H) Extracellular glucose concentrations in the dialysis buffer from mPFC samples at on every occasion point (n = 5 to 6 mice per team). Glimpse Supplies and Recommendations for detailed statistical analyses. (I) Plasma glucose concentrations (n = 6 to 7 mice per team). The main dimension used to be performed after 16 hours of fasting, and the second blood sequence used to be performed 30 min after eating 0.05 g of carbohydrates. (J) Fasting plasma insulin ranges (n = 5 to 6 mice per team). (D and H to J) Starch +/+ team used to be frail as a preserve an eye on for put up hoc Dunnett’s take a look at. All recordsdata are presented as components ± SEM. *P < 0.05.

We subsequent speculated that the irregular vascular endothelial cells and astrocytes seen in G × E mice can even alter glucose uptake from the plasma into the brain parenchyma. Extracellular glucose concentrations in the brain parenchyma were measured below three instances: (i) fasting, (ii) 1 hour after feeding, and (iii) 2 hours after feeding. We first measured a baseline glucose focus in the mPFC in starch-fed WT mice after starvation for 16 hours (Fig. 4H). This focus increased vastly within an hour after starch feeding and diminished shut to baseline 2 hours after feeding. Sucrose-fed WT mice and starch-fed Glo1/+ mice had identical baseline glucose concentrations, identical will enhance 1 hour after starch or sucrose feeding, and identical decreases abet to baseline 2 hours after feeding. In difference, sucrose-fed Glo1/+ mice had each vastly diminished basal glucose concentrations in the mPFC and no obvious lift in glucose concentrations after sucrose feeding. We measured vascular diameter and expression of glucose transporter 1, the foremost glucose transporter expressed in vascular endothelial cells and astrocytes, and seen no differences among the four groups (fig. S6). Furthermore, no differences were detected in plasma glucose after starvation and feeding or in fasting plasma insulin ranges among the four groups (Fig. 4, I and J), indicating that the decrease parenchymal glucose concentrations in G × E mice are ensuing from reduced uptake all the contrivance in which thru the BBB in set of dysregulation of plasma glucose or insulin signaling.

Defending effects of chronic low-dose aspirin against behavioral abnormalities and angiopathy

Outdated studies enjoy shown that adjunct nonsteroidal anti-inflammatory drug (NSAID) remedy can pork up psychiatric disorder ratings (40, 41). Aspirin, an NSAID, is automatically frail for the prevention and alleviation of vascular-associated adverse events associated with high blood stress, ischemia, and cardiovascular illnesses (42, 43). Here, we examined whether aspirin remedy can provide protection to against the plot of psychiatric phenotypes in G × E mice (Figs. 1A and 5, A to D, and fig. S7, A to I). Low-dose aspirin (1 mg/kg per day) fully prevented hyperlocomotor process, deficits in PPI, working memory, and grooming length (Fig. 5, A to D) and partly prevented irregular enhancement of dopamine starting up after methamphetamine administration and reduced PV expression among G × E mice (fig. S7, D to F) but didn’t pork up acoustic startle responses, nest-constructing and elevated plus maze ratings, and irregular astrocyte activation (fig. S7, A to C and G to I). These enhancements were accompanied by a decrease in endothelial fibrin accumulation (Fig. 5, E and F) and a partial restoration of glucose intake into the brain parenchyma (Fig. 5G). Collectively, these outcomes imply that aspirin remedy in G × E mice vastly improves angiopathy and brain glucose availability, contributing to the prevention of numerous behavioral abnormalities.

Fig. 5.

Defending effects of low-dose aspirin in G × E mice.

(A to D) Outcomes of behavioral tests performed to purchase into consideration the effects of aspirin remedy (n = 12 to 21 mice per team). (A) Quantification of spontaneous locomotor process in the OF. (B) PPI at 70 dB. (C) OLT of working memory. (D) Quantification of nest-constructing abilities over 8 hours. (E) Immunohistochemical photos of fibrin and the endothelial cell marker tomato lectin. (F) Size of the gap of fibrin immunoexpression in (E). The mean depth of the fibrin-immunopositive space of the complete image used to be measured for each section (n = 3 mice per team). (G) Extracellular glucose concentrations in the dialysis buffer at on every occasion point (n = 4 to 6 mice per team). Post hoc Tukey’s more than one comparisons take a look at of groups at on every occasion point, ###P < 0.001, ##P < 0.01, and #P < 0.05 for Starch +/+ versus Suc Glo1/+; $$$P < 0.001 and $P < 0.05 for Starch +/+ Aspi versus Suc Glo1/+; &P < 0.05 for Starch +/+ versus Suc Glo1/+ Asp. All recordsdata are presented as components ± SEM. P < 0.001, P < 0.01, and *P < 0.05.

Angiopathy in postmortem brains of patients with psychiatric disorders

Last, we compared immunostains of brain slices from healthy controls and patients with SZ or BD to examine whether these patients exhibit angiopathic fibrin accumulation in vascular endothelial cells. Similar to our findings in G × E mice, we found significantly elevated fibrin accumulation in the vascular endothelium of brain slices randomly collected from patients with SZ and BD (Fig. 6, A to C). Therefore, fibrin-related angiopathy and vascular damage may be a novel and common phenotype of psychiatric illness contributing to disease progression.

Fig. 6.

Angiopathy in postmortem brains from individuals with psychiatric disorders.

(A) Representative immunohistochemical fibrin images in the BA9 region of postmortem brains from controls and patients with schizophrenia (SZ) or bipolar disorder (BD). (B) Measurement of the area of fibrin immunoexpression in (A). The mean intensity of the fibrin-immunopositive area of the entire image was measured for each section. (C) Representative immunohistochemical images of fibrin (magenta) and the endothelial cell marker tomato lectin (green) in postmortem brains from a patient with SZ. Fibrin-positive areas are merged with areas of vascular endothelial cell marker expression. (D) Diagram describing the hypothesis proposed to explain functional and behavioral abnormalities in G × E mice (see Discussion for details). All data are presented as means ± SEM.

DISCUSSION

Using a mouse model, we demonstrated that high-dietary sucrose consumption during adolescence is a potential risk factor for the development of behavioral phenotypes associated with psychiatric illnesses, such as SZ and BD. These behavioral phenotypes include impaired sensory gating, dysfunctions in PV interneurons and working memory, hyperactivity, and increased basal and stimulus-evoked striatal dopamine release. Second, we identified endothelial fibrin accumulation (“angiopathy”) in both our mouse model and in randomly collected postmortem brains of patients with SZ or BD. We also observed that glucose intake from the plasma into the brain parenchyma was impaired in G × E mouse model. Chronic low-dose aspirin treatment prevented fibrin deposition in the capillaries, improved glucose transport, and reversed several behavioral phenotypes in G × E mice, suggesting angiopathy as a seminal pathogenic event in mental illness. We summarized our findings in table S1.

In Fig. 6D, we propose a possible model summarizing our results. A high-sucrose diet causes AGE accumulation in microglia and endothelial cells, which express low amounts of GLO1 (Fig. 3, A to H, and fig. S5, A to E). Astrocytes are less sensitive to this diet in WT animals because they express high amounts of GLO1 to relieve glycation stress (fig. S4, A to G). However, the reduced GLO1 in Glo1/+ mice causes astrocytes to become activated by a high-sucrose diet (Fig. 3, I to K). This activation may be caused directly by glycation stress in astrocytes or may occur as a consequence of increased microglial and endothelial AGEs. Astrocytic activation further increases microglial and endothelial AGEs in a positive feedback loop (7). Increased microglial and endothelial AGEs and astrocytic activation may induce fibrin accumulation and damage to the brain vasculature (34) (Fig. 4, C to G). This results in decreased glucose import into the brain and dopaminergic and PV neuron dysfunction, which in turn cause psychosis-associated behavioral defects (Figs. 1, B to H, 2, and 4H).

Notably, administration of the NSAID aspirin, which inhibits inflammation and oxidative stress, protected against the emergence of angiopathy as evidenced by reduced fibrin accumulation, partially restored parenchymal glucose concentrations, and prevention of several psychiatric-related phenotypes (Fig. 5 and fig. S7, D to F). The increase in coagulation factor V and the accumulation of fibrin in endothelial cells in G × E mice indicate the presence of some vascular damage (Fig. 4, A to G) (44). Aspirin may prevent this damage through its antiplatelet coagulation and anti-inflammatory properties. Since oxidative stress and chronic inflammation are also common features of psychiatric disorders, aspirin may provide a protective effect against the etiology of these diseases as well (8, 11, 45, 46). We have tried to quantify reactive oxygen species (ROSs) in our experimental mice but, thus far, have not been able to see differences among the four groups (fig. S8), so a more sensitive detection method such as fluorescence resonance energy transfer probe for ROS detection may be required.

Sucrose consists of glucose and fructose, and when these sugars are ingested at the same time, we find that they generate AGEs in different cell types. Glucose metabolism–derived AGEs, which can be detected using α-AGE polyclonal antibody, accumulated primarily in vascular endothelial cells (Fig. 3, A to H), while methylglyoxal-derived AGEs, recognized by the α-AGE-4 monoclonal antibody, accumulated specifically in microglia (fig. S5, A to E). Our results suggest that these two cell types may import and metabolize these two sugars differently, but more detailed validation, such as one-cell metabolomic analysis, is required to validate this idea.

Maturation of PV neurons including mPFC region at the circuit level occurs in late adolescence in humans, and malfunction of their circuit formation is thought to be the cause of core symptoms of psychiatric disorders (4749). Robust PV neuron function is required for PPI, working memory, amphetamine-induced hyperlocomotion, dopamine (or 3,4-dihydroxyphenylacetic acid) release regulation, and gamma oscillation generation, all of which are considered core symptoms of psychiatric disorders (27). The PV neuron dysfunction in G × E mice may be caused by the unique properties of these cells. PV-expressing interneurons are the fast-spiking neuron subtype exhibiting a lower input resistance and higher-amplitude rapid after-hyperpolarization than several projection neurons, and the combination of these properties generates higher frequency action potentials compared with those by other neuron types. This frequency of high action potentials in PV neurons can be exploited by their fast spike phenotype to produce fast oscillations (30 to 100 Hz) in the gamma band of neural oscillations (50). To maintain this rapid spiking property, PV neurons require high energy expenditure as evidenced by mitochondrial and cytochrome c oxidase enrichment (51). Therefore, reduced glucose within the brain parenchyma may preferentially disturb PV neuron function, resulting in reduced PV expression. Reduced PV neuron number has been repeatedly reported in postmortem brains of patients with psychiatric disorders such as SZ and BP, and this reduction is proposed to be caused by a decrease in PV mRNA or protein expression in these cells, rather than by a loss of PV neurons themselves (2326). Since PV is a Ca2+-binding protein, proper regulation of the expression of this protein is considered to be important for maintaining the plasticity of PV neurons. For example, in PV neurons of the hippocampus and mPFC, the expression of PV protein changes after contextual fear conditioning, exposure to rich environments, and pharmacogenetic activation of PV neurons, suggesting that PV expression and plasticity are correlated (26, 52). Furthermore, it is known that the inhibitory activity of PV neurons is critical under environmental stress to prevent a sequela of excessive excitatory activity such as oxidative stress and inflammation (51). Pathogenic processes might more readily occur during the critical adolescent prodromal period, wherein PV neurons may have increased vulnerability to environmental stresses because of delayed maturation, which is another feature of PV neurons (53).

In the present study, we identified capillary angiopathy in both G × E mice and postmortem brains of patients with SZ and BD (Figs. 4, C to G, and 6, A to C). In our G × E mice, angiopathy was caused by the high AGE production capacity of sucrose combined with a GLO1 deficiency. Since high sugar consumption has been associated with SZ and BD and decreased GLO1 expression and function have been seen in SZ and BD, similar interactions may be associated with some cases of psychiatric diseases. However, various other environmental stresses and genetic conditions may also converge to induce angiopathy. Several studies have reported that stressors, such as social defeat, isolation, and viral infection, induce vascular defects (5456). These stressors are also risk factors for SZ and BD and induce PV neuron hypofunction (57), suggesting that angiopathy may be a common trigger for psychiatric phenotypes.

MATERIALS AND METHODS

Experimental design (related to Fig. 1A)

After weaning (P21), WT and Glo1 heterozygous mutant mice were fed either a starch diet (control) or a sucrose diet (experimental). Diets were equal in total calories and proportions of calories from carbohydrates, lipids, and proteins. The behavioral test battery was administered starting at 2.5 months of age (top panel). Middle panel shows the macronutrient composition of the two diets. We used Glo1 heterozygous mice (or Disc1 heterozygous mice) to mimic patients with psychiatric disorders who exhibit decreased GLO1 activity or expression (or decreased DISC1 expression), whereas high sucrose intake was used as an environmental risk factor (bottom panel). We investigated four groups of mice: WT, starch-fed mice (Starch +/+); Glo1 (or Disc1) heterozygous, starch-fed mice [Starch Glo1/+ (or Starch Disc1/+)]; WT, sucrose-fed mice (Suc +/+); and Glo1 (or Disc1) heterozygous, sucrose-fed mice [Suc Glo1/+ (or Suc Disc1/+)].

Animals

All experimental procedures were approved by the Animal Experimentation Ethics Committee of the Tokyo Metropolitan Institute of Medical Science (49040). All mice were maintained under a 12: 12-hour light:dark cycle (lights on at 8: 00 a.m.) with free access to the indicated diet. All efforts were made to minimize the number of animals used and their suffering. Glo1 knockout mice were generated as described previously (58, 59). Briefly, Glo1-trapped ES cell lines from the International Gene Trap Consortium were used for the generation of three founder mice, which were then backcrossed to C57BL/6 mice. In the manuscript, trapped Glo1 is referred to as Glo1. Disc1-LI (locus-impairment) heterozygous mutant mice were generated as previously described (17). In the manuscript, Disc1-LI is referred to as Disc1. Alternatively, mice were backcrossed to GFAP-GFP mice to monitor astrocyte activation (32, 33). Male mice were exclusively used in the behavioral tests, whereas mice of both sexes were used in histological, biochemical, and physiological experiments.

Diet preparation

The two diets used in the present study were newly created in collaboration with Oriental Yeast Co. Ltd. (Tokyo, Japan). We named the sucrose diet HSD-70 (#OYC 2405100) and the starch diet HCD-70 (#OYC 2405000) (table S4). They contained the same caloric proportions of carbohydrate, fat, and protein; however, all carbohydrate calories are derived from either starch or sucrose.

Drug preparation

Aripiprazole was dissolved in acetic acid and diluted to 3.5 mg/liter in water for administration at 0.5 mg/kg per day. The final acetic acid concentration in the drinking water was 0.7%. Aspirin was dissolved in ethanol and diluted to 70 mg/liter in water for administration at 1 mg/kg per day. The final ethanol concentration in the drinking water was 0.15%. The daily dose was based on the measurement of mean water consumption daily in all sucrose-fed Glo1 heterozygous mice and starch-fed WT mice (fig. S1, D and E). There were no between-group differences in terms of aripiprazole consumption (Student’s t test, Starch +/+ and Suc Glo1/+: P = 0.86) or aspirin-containing water (Student’s t test, Starch +/+ and Suc Glo1/+: P = 0.61).

Behavioral tests

Mice were habituated in the behavioral room for >30 min before each take a look at. Behavioral tests were performed in the following sequence of increasing stress: elevated plus maze, grooming, nest constructing, OF, object space, social interaction, and PPI. All take a look at apparatuses were cleaned using 70% ethanol and water between trials, and the following take a look at session used to be started most keen after the ethanol vapor odors had disappeared and the apparatuses had dried.

The elevated plus maze (EPM-04M, Muromachi, Japan) consisted of two opposing starting up fingers (297 mm by 54 mm) and two closed fingers (300 mm by 60 mm by 150 mm) extending from a central platform (60 mm by 60 mm). Your complete apparatus used to be elevated 400 mm above the ground. Every mouse used to be placed on the central platform facing a closed arm and allowed to freely in discovering the maze for 10 min. Arm entry used to be outlined as the entry of all four paws into the arm. The time spent in the starting up fingers over 10 min used to be recorded as an index of bid alarm.

For the self-grooming take a look at, all mice housed in the identical dwelling cage were moved into a recent cage for 10 min. Every mouse used to be then in my view placed in a mature mouse dwelling cage (31 cm by 16.5 cm by 14 cm) illuminated at ~200 lux. After a 10-min habituation length, each mouse used to be scored for cumulative time spent grooming all body areas (60) over 10 min using a stopwatch. Self-grooming habits is conserved all the contrivance in which thru species and is indicative of certain pathological instances or components. In folk, as an illustration, self-grooming will enhance in the course of annoying instances and in certain psychiatric concerns (60).

For the nest-constructing take a look at, 200 g of corncob used to be spread all the contrivance in which thru the bottom of every cage for bedding, and a square-formed portion of cotton used to be placed in the cage center as raw cloth for the nest. Every mouse used to be in my view placed in the cage for 8 hours. Photography of the constructed nest were bought every 2 hours, and the nest-constructing course of used to be evaluated by measuring the share of loose cotton as follows: one point for 25% weight [weight % (wt %)] loosened, two aspects for 50 wt % loosened, three aspects for 75 wt % loosened, and four aspects for 100 wt % loosened. After 8 hours, we checked the form of the nest and added one point if the mice had carried out a nest with a chook’s nest-take care of form. The temperature of the room used to be maintained at 25°C and illumination at 150 to 180 lux in the course of nest constructing. Nest-constructing habits is a hallmark of total smartly-being in mice (61), whereas miserable nest constructing is a hallmark of psychological or physiological abnormalities (62, 63).

For the OF take a look at, each mouse used to be placed in the center of the apparatus (40 cm by 40 cm by 40 cm; 150 to 180 lux illumination) and allowed to switch freely for 10 min. The habits of every mouse used to be monitored using a fee-coupled tool digicam mounted on the ceiling above the OF. The total distance traveled (in centimeters) used to be measured using CompACT VAS instrument (Muromachi).

For the OLT of working memory (64), mice first explored the empty OF box, after which, two identical objects A and B (two 500-ml polyethylene terephthalate bottles stuffed with blue-colored water) were placed in two corners 5 cm from the wall. After a 10-min exploration/discovering out length, the mice were returned to their dwelling cage for 5 min, and object A used to be moved to a recent nook (object A′). The animals were then placed abet in the OF box and allowed to in discovering for 5 min. The time spent exploring A′ and B were measured to calculate a discrimination index representing working memory in maintaining with the following equation: discrimination index = (recent object A′ exploration time − acquainted object B exploration time)/(recent object A′ + acquainted object B exploration times). The OLT used to be performed below illumination at 10 to 15 lux.

The social interaction take a look at used to be conducted as previously described (65) using a specialized sociability apparatus (SC-03M, Muromachi). The time spent sniffing a recent stimulus mouse or object used to be manually scored from videos recorded using an overhead colour USB digicam (aa000080a02, Iroiro Dwelling). Stimulus mice (129Sv/SvJcl stress) age- and sex-matched to envision mice were habituated to the apparatus and to the enclosure cup for 30 min/day for 2 days before trying out. The place of dwelling (left or exquisite) of the unconventional object and recent mouse within an enclosure used to be alternated all the contrivance in which thru take a look at topics. The take a look at mouse used to be allowed to acclimate to the apparatus for a complete of 20 min before the sociability take a look at—the first 10 min in the central chamber with the doors closed after which 10 min in the empty enviornment with the doors starting up. The take a look at discipline used to be temporarily confined to the center chamber, whereas a recent stimulus mouse in an enclosure cup used to be placed on one in all the facet chamber, and one other empty enclosure cup (recent object) used to be placed on the assorted facet of the chamber. The take a look at discipline used to be allowed to plot the unconventional object or mouse freely for 10 min. The time spent interacting with the stimulus mouse versus the unconventional object used to be calculated as an index of sociability.

The SR-LAB-Startle Response Map (San Diego Instruments) used to be frail to detect acoustic startle reflexes and PPI. Startle responses were measured using five stimulus intensities (80, 90, 100, 110, and 120 dB) delivered 10 times each for 40 ms over a white noise background (65 dB). The stimuli were presented in quasi-random order at random intertrial intervals (10 to 20 s). Within the PPI session, mice were exposed to two stimulus patterns: (i) a startle stimulus by myself (120 dB, 40 ms) with no prepulse stimulus and (ii) a startle stimulus (120 dB, 40 ms) following a prepulse stimulus (70 dB for 20 ms; lead time, 100 ms). Every trial used to be repeated 10 times in quasi-random order at random intertrial intervals (10 to 20 s). PPI used to be outlined as the p.c decline in startle response thanks to prepulse stimuli in maintaining with the following equation: 100 − [(120 dB startle amplitude after any prepulse)/(120 dB startle amplitude only)] × 100. A recent accelerometer used to be frail for the experiments with Disc1 knockout mice.

Immunohistochemistry

Following transcardial perfusion with phosphate-buffered saline (PBS) and 4% paraformaldehyde (PFA), complete brains were mute, postfixed at 4°C overnight, and cryoprotected in 20% sucrose at 4°C overnight. Serial coronal sections (50 μm) were then cut using a cryostat (CM3050 S; Leica Microsystems). The antigens in the tissues were reactivated by heating in HistoVT One solution (Nacalai Tesque) for 30 min at 70°C using a water tub. Sections were permeabilized with 0.2% Triton X-100 and 1% Block Ace (DS Pharma Biomedical) in PBS for 30 min at room temperature, following which they were incubated overnight with the indicated foremost antibodies at room temperature. For immunohistochemistry of postmortem human brain tissues, paraffin blocks including BA9 (a place of frontal cortex) were sliced into 10-μm sections, deparaffinized with xylene, and rehydrated with decreasing concentrations of ethanol in water. Antigens were reactivated by heating in HistoVT One solution for 30 min at 90°C using a water tub. Sections were treated with TrueBlack Lipofuscin Autofluorescence Quencher (Biotium Inc.) for 30 s at room temperature and blocked with 1% Block Ace (DS Pharma Biomedical) in PBS for 30 min at room temperature. Thereafter, the mouse and human brain sections were subjected to the identical immunostaining procedures. The following foremost antibodies were diluted in PBS containing 0.4% Block Ace: goat anti-PV (1: 2000; Frontier Institute, PV-Roam-Af860), mouse anti-ALDH1L1 (1: 200; Rockland, 600-101-HB6S), fluorescein isothiocyanate–conjugated tomato lectin (1: 200; VECTOR, FL-1171), chick anti-GFP (1: 500; Abcam, ab13970), goat anti-IBA1 (1: 100; Abcam, ab48004), mouse anti-NeuN (1: 500; Millipore, MAB377), rabbit anti-AGE (1: 2000; Abcam, ab23722), rabbit anti-IBA1 (1: 300; Wako, WDJ3047), rat anti-CD68 (1: 500; Abcam, ab53444), and rabbit anti-fibrin (1: 500; Dako, A0080). Thereafter, sections were washed thrice with PBS containing 0.05% Tween 20, incubated for 2 hours with fluorochrome-conjugated secondary antibodies in PBS containing 0.4% Block Ace, and washed an additional thrice in PBS containing 0.4% Block Ace. For enhanced horseradish peroxidase (HRP) immunostaining, samples were treated with 3% H2O2 in PBS for 20 min following the reactivation step to quench endogenous peroxidase process and washed in PBS. Sections were incubated with rabbit anti-GLO1 (1: 1500; Novusbio, NBP2-75514) and/or mouse anti–AGE-4 (1: 400; Trans Genic Inc., 14B5), followed by incubation with anti–immunoglobulin G (IgG) antibodies conjugated to biotin (1: 200; VECTOR). After washing as described for assorted secondary antibodies, sections were incubated with streptavidin-conjugated HRP (1: 200; Jackson ImmunoResearch) for 120 min and washed thrice with PBS containing 0.05% Tween 20. The TSA Plus Fluorescence Map (PerkinElmer) used to be frail to detect HRP process. All preparations were counterstained with 4′,6-diamidino-2-phenylindole (Nacalai Tesque) to order cell nuclei, washed three additional times, mounted in Permaflow (Thermo Fisher Scientific), and seen using a FluoView FV3000 Confocal Laser Scanning Microscope (Olympus).

Picture analysis

Except in any other case renowned, all image analyses were performed using ImageJ version 2.0.0-rc-59/1.51n, and photos were first binarized. PV-obvious cells with a threshold exceeding 30 were counted.

To measure the areas immunopositive for AGE, fibrin, and AGE-4, the threshold settings were applied to form certain most keen the exquisite immunopositive space used to be properly selected, and the stained space of the complete masks used to be measured. For measuring the fluorescence depth of GFP and IBA1, after surroundings the place of curiosity to purchase most keen purpose immunoreactive cells, the depth of >5 cells in each image used to be measured. To rely microglial cells containing a CD68-obvious space exceeding 20 μm2, the threshold used to be dwelling to purchase most keen the CD68-obvious space. Colocalization analysis of two assorted fluorescence markers used to be performed using the Fiji slip-in Coloc 2 with the default settings. We adopted Pearson’s R fee beneath the threshold to judge the strength of colocalization: R ≥ 0.7, tough; 0.7 > R ≥ 0.4, life like; 0.4 > R ≥ 0.2, mature; and R < 0.2, none or very mature.

Immunoblotting

Extracts from mouse hippocampi were homogenized in lysis buffer containing 40 mM tris shocking, 0.4% SDS, 0.01 M EDTA (pH 8.0), 8 M urea, and 1 mM phenylmethylsulfonyl fluoride. The total lysate protein squawk material used to be quantified using a DC Protein Assay Equipment (Bio-Rad). Total protein (30 μg per gel lane) used to be separated using SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore). Membranes were blocked with tris-buffered saline with Tween 20 (TBST) buffer [137 mM NaCl, 2.7 mM KCl, and 25 mM tris (pH 8.0)] including 0.2% Triton X-100 and 5% bovine serum albumin (BSA) for 30 min at room temperature with slack shaking, followed by incubation overnight with foremost antibodies in TBST including 2% BSA at 4°C. The main antibodies frail were rabbit anti-GLO1 (1: 1000; Santa Cruz Biotechnology, sc-67351), mouse anti-PV (1: 1000; Swant, PV-235), rabbit anti–glucose transporter 1 (1: 1000; Glut1; Frontier Institute, Af1020), and mouse anti-tubulin (1: 10,000; Santa Cruz Biotechnology, sc-32293). After washing thrice with TBST, membranes were incubated with the secondary antibody (1: 2000; HRP-conjugated anti-mouse or anti-rabbit IgG antibody, GE Healthcare) in TBST plus 2% BSA. After washing thrice with TBST, blots were processed for chemiluminescence using traditional protocols (ECL Prime Western Blotting Detection Regent #RPN2236, GE Healthcare), and alerts were detected using a LAS 4000 Imager (Fujifilm).

Microdialysis

We frail an in vivo microdialysis system for the dimension of extracellular dopamine focus (66) and sequence of brain parenchymal dialysate (Fig. 1, H and J, and fig. S7D). After anesthesia by intraperitoneal injection of ketamine (80 mg/kg)/xylazine (16 mg/kg), mice were fastened in a stereotaxic apparatus (Narishige), and a microdialysis recordsdata cannula (CXG-8, Eicom) used to be implanted in the mPFC [antero-posterior (AP), +1.8 mm; medio-lateral (ML), ±0.15 mm; dorso-ventral (DV), −1.5 mm from bregma] or NAc (AP, +1.5 mm; ML, ±0.6 mm; DV, −3.5 mm from bregma). After restoration for no longer decrease than 10 days, a microdialysis probe (CX-I-8-01 for the mPFC and CX-I-8-02 for NAc; Eicom) used to be inserted thru the recordsdata cannula. Following insertion, the probe used to be connected to a syringe pump, and perfusion used to be performed at 2 μl/min for NAc and nil.5 μl/min for mPFC using Ringer’s solution (147 mM NaCl, 4 mM KCl, and just a few.3 mM CaCl2). Dialysate samples were mute every 10 min and automatically loaded onto an HTEC-500EPS HPLC unit (Eicom). Constant serotonin (5-hydroxytryptamine) focus in three consecutive sequence intervals used to be first confirmed to rule out blood contamination before initiating the measurements of dopamine focus or sequence of parenchymal dialysates. Analytes were then separated on an affinity column (PP-ODS III, Eicom), and compounds were subjected to redox reactions within an electrochemical detection unit (amperometric DC mode; applied doable fluctuate, 450 mV). The ensuing chromatograms were analyzed using an EPC-500 recordsdata processor (Eicom), and staunch sample concentrations were computed on the basis of the height heights obtained using 0.01, 0.1, and 1 pg of dopamine in traditional solution (Sigma-Aldrich). The areas of the microdialysis probes were histologically confirmed.

For glucose measurements in Figs. 4H and 5G, we mute dialysates from the mPFC for 1 hour after 16 hours of fasting, followed by 0 to 1 hour after eating 0.05 g of carbohydrates (starch or sucrose), and 1 to 2 hours after eating 0.05 g of carbohydrates (starch or sucrose).

EEG recordings

For behavioral and video/EEG monitoring, mice were anesthetized by an intraperitoneal injection of ketamine (80 mg/kg)/xylazine (16 mg/kg), fastened in a stereotaxic apparatus (Narishige, Japan), and implanted with EEG and electromyography (EMG) electrodes. The EEG electrodes were gold-covered stainless-steel screws (SUS303) soldered with lead wires (ANE-0190, Adler’s Nest, Japan) implanted epidurally over the left frontal cortex (AP, 1 mm; ML, 1 mm) and the bilateral parietal cortex (AP, −2 mm; ML, ±2 mm). All wires were soldered to a multichannel electrical connector (R833-83-006-001, TOKIWA SHOKO, Japan). The left parietal cortex electrode used to be frail as a reference for monitoring the frontal cortex EEG. The EMG electrodes were lead wires placed bilaterally into the trapezius muscle. Following restoration for no longer decrease than 10 days, EEG/EMG alerts were amplified and band-disappear filtered (EEG: 1.5 to 1000 Hz; EGM: 15 to 3000 Hz) using a MEG-6116 system (NIHON KOHDEN), digitized at a sampling fee of 200 Hz, recorded using a recordsdata acquisition system (PowerLab 8/30, ADInstruments), and analyzed using LabChart Tool (ADInstruments). Behavioral actions were recorded using a USB digicam (aa000080a02, Iroiro Dwelling, Japan). Behavioral and electrophysiological responses to a recent object (an empty 100-ml DURAN bin) were recorded in an OF chamber (20 cm by 20 cm by 26 cm). The recent object used to be placed in a single nook of the OF chamber to induce exploration. The 30 s preceding the first contact with the unconventional object used to be analyzed for object recognition (“object process”). For EEG monitoring in the home cage, mice were first habituated for 8 hours. Dwelling cage EEG recordsdata were then bought for 2 min after awaking as confirmed by obvious EMG alerts and rush photos from an offline video digicam analysis (“dwelling cage process”). All recordings were converted into energy spectra using a quick Fourier transform (FFT) algorithm with a 5-s Hann cosine-bell window and 50% overlap between successive window measurements. All FFTs were maintained at 1024 aspects to develop 0.512-Hz decision. The total stamp amplitude or energy (V2) in each 5-s length used to be measured as the energy magnitude at each frequency. The mean of the grouped energy spectra used to be calculated over the following frequency ranges: 1 to 4 Hz (delta), 5 to 10 Hz (theta), 30 to 45 Hz (low gamma), and 55 to 80 Hz (high gamma). The energy values detected at each frequency fluctuate for 30 s were additional averaged over 30 s of total EEG energy using the mean values to tell doable noise. These analyses were performed using personalized instrument written in MATLAB (R2019b; MathWorks).

Transcriptome analysis

Three self sustaining total RNA samples from each team were combined and purified using an RNeasy Mini Equipment (Qiagen). RNA quality used to be assessed using a 2100 Bioanalyzer (Agilent Applied sciences). Cy3-labeled complementary RNA used to be ready using a Low Input Fleet Amp Labeling Equipment (Agilent Applied sciences) in maintaining with the manufacturer’s protocol. Samples were hybridized to the SurePrint G3 Mouse Gene Expression v2 Microarray (G4852B; Agilent Applied sciences). Thereafter, the array used to be washed and scanned using the SureScan microarray scanner (Agilent Applied sciences). Microarray photos were analyzed using the Characteristic Extraction instrument with default settings for all parameters (Agilent Applied sciences). Recordsdata from each microarray analysis were normalized by shift to the 75th percentile without baseline transformation. Microarray outcomes were deposited in the Gene Expression Omnibus database below the accession quantity GSE141829.

Insulin and glucose measurements

Blood plasma used to be mute from the mouse cheek as described by Golde et al. (67). Plasma glucose focus used to be measured using a Precision-Neo blood glucose meter (#71386-80, Abbott, Japan), plasma insulin focus used to be measured using an enzyme-linked immunosorbent assay (ELISA) equipment (#M1102, MORINAGA), and glucose focus in the dialysate samples used to be measured using a undeniable ELISA equipment (#ab65333, Abcam), all in maintaining with the producers’ instructions. Recordsdata were mute on a microplate reader (Varioskan, Thermo Fisher Scientific).

Human postmortem brain tissue sequence

Postmortem brain tissues from sufferers with SZ and BD were obtained from the Fukushima Mind Monetary institution at the Department of Neuropsychiatry, Fukushima Medical College. Postmortem brain tissues from preserve an eye on folk were obtained from the Share of Pathology, Fukushima Medical College Health facility. The utilization of postmortem human brain tissues in the latest in discovering used to be authorized by the Ethics Committee of Fukushima Medical College (no. 1685) and Tokyo Metropolitan Institute of Medical Science (no. 18-20) and complied with the Declaration of Helsinki and its later amendments. All procedures were conducted with the urged written consent of the following of family. Detailed demographic recordsdata of the 10 sufferers with SZ, 9 sufferers with BD, and the 12 age- and sex-matched preserve an eye on folk is equipped in table S5. No between-team differences were seen in the case of sex (Fisher’s real take a look at, Ctrl and SZ: P = 1.00, Ctrl and BD: P = 0.40), age (Student’s t take a look at, Ctrl and SZ: P = 0.69; Welch’s t take a look at, Ctrl and BD: P = 0.66), postmortem interval (Student’s t take a look at, Ctrl and SZ: P = 0.89, Ctrl and BD: P = 0.98), or a history of diabetes mellitus (Fisher’s real take a look at, Ctrl and SZ: P = 0.59, Ctrl and BD: P = 0.59). Every affected person with SZ and BD fulfilled the diagnostic criteria established by the American Psychiatric Affiliation (Diagnostic and Statistical E-book of Mental Disorders, DSM-IV) and didn’t enjoy a past history of assorted neurological concerns or substance abuse. Furthermore, no longer one in all the preserve an eye on folk had any file of psychological concerns, neurological concerns, or substance abuse.

ROS detection

Dihydroethidium (DHE) is oxidized by superoxide and numerous reactive species to form a precipitate that emits pink fluorescence, whose depth can even unprejudiced additionally be frail to measure the extent of cell oxidative stress. Animals were quickly perfused with ice-chilly PBS, the decapitated brains were embedded in OCT compound (Sakura Finetek, Japan), and 10-μm-thick coronary sections were ready in a cryostat. These sections were incubated with phosphate buffer containing 100 μM diethylenetriamine pentaacetic acid (PB/DTPA) for 10 min after which incubated with PB/DTPA containing 10 μM DHE (Invitrogen) for 20 min. After washing with PB/DTPA twice, sections were fastened with 4% PFA for 15 min. As soon as washed with PBS, PBS containing 1 μM TO-PRO 3 (Thermo Fisher Scientific) used to be applied to each section followed by three washes with PBS. Then, sections were sealed with Permaflow (Thermo Fisher Scientific) and seen using a FluoView FV3000 confocal laser scanning microscope (Olympus). For DHE detection, we frail the following filter dwelling: excitation, 405 nm and emission, 600 to 650 nm.

Statistical analyses

Statistical differences among ≥4 groups were certain using one-contrivance analysis of variance (ANOVA), two-contrivance ANOVA, three-contrivance ANOVA, or repeated measures ANOVA, followed by the Bonferroni more than one comparison take a look at or Tukey-Kramer take a look at as a put up hoc take a look at, as summarized in table S6. We frail the identical animals for all outcomes coming from Starch +/+ and Suc Glo1/+ groups in maintaining with the 3R rule. Recordsdata were mute at the identical time regardless of whether the drug used to be administered or no longer. The sequence of animals frail for each experiment/analysis can even unprejudiced additionally be found in each figure fable. Detailed descriptions of statistical analyses in Figs. 1 (H and J) and 4H are shown beneath. In Fig. 1H, put up hoc Dunnett’s more than one comparisons take a look at for groups used to be performed at order time aspects (0 and 40 min), ###P < 0.001 for Suc Glo1/+ versus Ctrl (Starch +/+), and $P < 0.05 for Starch Glo1/+ versus Ctrl (Starch +/+). In Fig. 1J, put up hoc Bonferroni’s more than one comparisons take a look at for groups used to be performed at order time aspects (0 and 40 min), ###P < 0.001 for Suc Glo1/+ versus Starch +/+, $$$P < 0.001 for Suc Glo1/+ versus Starch +/+ Aripi, and &&&P < 0.001 for Suc Glo1/+ versus Suc Glo1/+ Aripi. In Fig. 4H, put up hoc Dunnett’s more than one comparisons take a look at for groups used to be performed at on every occasion point, ###P < 0.001 and ##P < 0.01 for Suc Glo1/+ versus Ctrl (Starch +/+).

Acknowledgments

We thank S. Ogikubo, Y. Matsumoto, H. Zhang, M. Murata, I. Nohara, Y. Shimada, E. Hama, N. Obata, M. Hatakenaka, and C. Ishida for contribution to experiments associated to this analysis. We also thank T. Kodama for explaining the procedures for microdialysis and encephalogram recording. We’re grateful to K. Tanaka for reviewing the in discovering. We acknowledge C. Watanabe and H. Onuma for coordinating the donations of postmortem brains. Furthermore, we thank H. Chiba for the preparation of postmortem brain samples. We order our gratitude to the households of the deceased folk for the donations of brain tissue and their time and effort devoted to the consent course of and interviews.

Funding: This work used to be supported by the Japan Society for the Promotion of Science (KAKENHI grants 18K14832 to S.H., 17K18395 and 19K08033 to H.M., 17K16408 to T.Ta., and 18H02537 and 18K19383 to H.O.); the Ichiro Kanehara Foundation, Japan Prize Foundation, and Takeda Science Foundation (to S.H.); the Naito Foundation (to H.M.); the Strategic Be taught Program for Mind Sciences from AMED (grant JP19dm0107107 to H.Y.); and a Grant-in-Aid for Scientific Be taught on Innovative Areas from the MEXT (JP16H06277 to H.Y.). This analysis used to be also supported by KAKENHI grant numbers 16H05380 (to M.A.), 18K06977 (to K.T.), 19H03589 (to M.I.), 18H02542 (to T.H.), and 18K15354 (to K.S.) as smartly as AMED grant numbers JP19dm0107088 (to M.I.) and JP21wm0425010 (to T.H.). This in discovering used to be also supported by The Kanae Foundation for the Promotion of Medical Science (to K.T.), The Uehara Memorial Foundation (to M.A.), and the Collaborative Be taught Program of Institute for Protein Be taught, Osaka College, ICR-21-3 (to T.H.).

Creator contributions: S.H. and H.O. designed the in discovering. S.H. performed and analyzed all experiments. H.M. assisted with the experimental construct of EEG recording and analysis of the outcomes and edited the manuscript. T.Ta. coordinated the EEG recording. Y.K., M.H., R.I., A.N., and H.Y. helped with the construct of experiments using human specimens and offered fastened human brain sections. T.S. performed recordsdata and image analysis. T.N. and S.S. offered GFAP-GFP mice. T.D. and T.M. generated and offered Glo1 knockout mice. K.T. and K.S. backcrossed Glo1 knockout mice to B6J mice for all experiments. Y.N. performed complementary DNA microarray analysis of gene expression. H.S. assisted with behavioral experiments. M.I., M.A., K.T., K.S., and M.M. offered significant solutions to this in discovering. T.To. and T.H. offered significant input on Disc1-LI mouse experiments. J.H. edited the manuscript. S.H. generated all figures and tables and wrote the manuscript. H.O. edited the manuscript and supervised this in discovering.

Competing interests: The authors bid that they have not any competing interests.

Recordsdata and materials availability: All recordsdata wished to purchase into consideration the conclusions in the paper are latest in the paper and/or the Supplementary Supplies. The accession quantity for transcriptome analysis recordsdata is equipped in Supplies and Recommendations.

Supplementary Supplies

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2 Comments

  1. You know that silly old fortune-cookie game where you add "in bed" to the end of the fortune? For example, "You will achieve great success… in bed".

    That, but for scientific articles and what you add to the end is "… in mice".