Differences in collateral vessel formation after experimental retinal vein occlusion in spontaneously hypertensive rats and wild-type rats.

Objective: Retinal vein occlusion (RVO) can lead to visual impairment, but the development of collateral vessels can sometimes mitigate significant damage. This study aimed to investigate the relationship between collateral vessels and hypertension, the most common underlying condition associated with RVO, by comparing spontaneously hypertensive rats (SHRs) and wild-type Wister rats (WWRs). We also examined the differences between WWRs and SHRs in terms of sphingosine 1-phosphate receptor 1 (S1PR1) expression and its product nitric oxide synthase 3 (NOS3) expression, which are involved in the formation of collateral vessels after vascular occlusion. Methods: Laser photocoagulation (PC) was used to occlude one randomly selected retinal vein in WWRs and SHRs, and the area surrounding the occluded vessel was examined using optical coherence tomography angiography. If reperfusion of the occluded vessel occurred within 2 weeks, the vessel was re-occluded repeatedly by PC. The number of eyes with successfully occluded vessels accompanied by collateral vessels was recorded. Then, WWRs and SHRs were divided into the following four groups: 1) control (no treatment), 2) vehicle (20% DMSO), 3) S1PR1 agonist (2 mg/mL SEW2871), and 4) S1PR1 antagonist (0.25 mg/mL VPC 23019) groups. The drugs were administered intravitreally in all groups except the control. The number of laser shots required for successful RVO was recorded. Histological evaluation and quantitative real-time PCR of S1PR1 and NOS3 were performed to elucidate the mechanisms underlying collateral vessel formation. Results: The proportion of eyes achieving successful vein occlusion was lower in SHRs (4/12 eyes, 33.3%) than in WWRs (8/10 eyes, 80%, p = 0.043). NOS3 expression at 6 h after PC was significantly higher in WWRs than in SHRs (p = 0.021). In WWRs treated with SEW2871, vein occlusion failed in 7 of 10 eyes (70%). The expression of NOS3 was significantly higher in the SEW2871 treatment group than in the untreated group (p < 0.001). Furthermore, NOS3 expression was significantly higher after SEW2871 treatment in WWRs than in SHRs (p = 0.011). Conclusion: In hypertensive environments, collateral vessels are less likely to develop, and S1PR1 may be involved in this phenomenon.

Histopathological findings of the nasopharynx-associated lymphoid tissue of pigs co-infected with porcine circovirus 2 and porcine reproductive and respiratory syndrome virus.

Porcine circovirus 2 (PCV2) causes porcine circovirus-associated disease, and co-infection with porcine reproductive and respiratory syndrome virus (PRRSV) severely affects the pig breeding industry. Both viruses target the macrophages in lymphoid tissues. Various porcine pathogens enter via the nasal cavity, and the nasopharynx-associated lymphoid tissue (NALT) acts as the mucosal immune system. However, the pathological analysis has not progressed. This study aimed to histologically examine the NALT of pigs with suspected PCV2 and PRRSV infections. Six pigs were subjected to necropsy, and their NALT, tonsils, and mesenteric lymph nodes were collected. Macrophages, lymphocytic depletion, multinucleated giant cells, intracytoplasmic inclusion bodies, and neutrophil infiltration increased in the NALT. In situ hybridization revealed positive signals for PCV2 in the NALT of all pigs and PRRSV in the NALT of three pigs. PCV2-positive macrophages were mainly identified in the follicles, whereas PRRSV-positive tissues were found primarily around the crypt and directly below the epithelium. Quantitative PCR revealed 108-1010 copies of PCV2 DNA/µL and 102-104 copies of PRRSV DNA/µL in the NALT. Therefore, both PCV2 and PRRSV were detected in the NALT of pigs. In conclusion, the infection and replication of both viruses in the NALT and tonsils may suppress host immunity and promote co-infection with other pathogens.

Detection of Swine Influenza A and Porcine Reproductive and Respiratory Syndrome Viruses in Nasopharynx-Associated Lymphoid Tissue.

Porcine respiratory disease complex, which is caused by a combination of pathogens, including swine influenza A virus (SIV) and porcine reproductive respiratory syndrome virus (PRRSV), results in significant economic losses in pig production systems. Nasopharynx-associated lymphoid tissue (NALT) plays an important role in the uptake of pathogens and defence of the nasal mucosa in rodents and humans. We characterized NALT M cells in pigs and detected SIV antigen and PRRSV nucleic acid in NALT using histopathological, immunohistochemical and in-situ hybridization analyses. All SIV- and PRRSV-positive cases examined had suppurative nasopharyngitis and pneumonia. M cells were detected by immunohistochemistry and the distribution of M cells showed an increase in the middle section of NALT. SIV antigen was detected in M cells and PRRSV nucleic acid was demonstrated in the cytoplasm of macrophages in NALT. We believe that SIV and PRRSV infection in the upper respiratory tract induces local immunosuppression and these results confirm that swine NALT is a location for virus replication and may be strongly associated with the development of pneumonia in pigs.

Time course of collateral vessel formation after retinal vein occlusion visualized by OCTA and elucidation of factors in their formation.

BACKGROUND: It is clinically recognized that collateral vessels can form after retinal vein occlusion (RVO) in some cases and these vessels can lead to spontaneous recovery of the pathological condition. In recent years, optical coherence tomography angiography (OCTA) has become a decisive clinical instrument. Unlike previous angiography tests, OCTA enables the non-invasive visualization of fundus vasculature without the need for administration of a contrast agent. However, it remains to be determined if OCTA depicts the 'true' histological status as several studies have reported artifacts in OCTA imaging. METHODS: We generated a laser-induced mouse RVO model, and evaluated the subsequent formation of collateral vessels in order to understand the mechanisms by which collateral vessels form using OCTA imaging, as well as molecular and histological assessments. RESULTS: We succeeded in visualizing the time course of collateral vessel formation in a mouse RVO model and confirmed the similarity in formation of collateral vessels only within the deep layer of the retina in both human and mouse. We hypothesized that sphingosine 1-phosphate receptor-1 (S1PR1) may play important roles via vascular shear stress linking vein occlusion and collateral vessel formation. Results from OCTA revealed that collateral vessels are increased in response to administration of a S1PR1 agonist in a mouse RVO model. Based on quantitative reverse transcription polymerase chain reaction (qRT-PCR), S1PR1 messenger ribonucleic acid (mRNA) levels in the whole retina peaked 6 h after photocoagulation in this model. Immunohistochemical staining of retinal flat mounts revealed that S1PR1 staining occurred along the laser-occluded blood vessels. CONCLUSION: We observed the temporal process of collateral vessel formation in a mouse RVO model and identified the relationship between S1PR1 and shear stress as one of the factors in collateral vessel formation in RVO.

Comprehensive behavioral analysis and quantification of brain free amino acids of C57BL/6J congenic mice carrying the 1473G allele in tryptophan hydroxylase-2.

AIM: Tryptophan hydroxylase 2 (Tph2) is a rate-limiting enzyme for the biosynthesis of 5-hydroxytryptamine (5-HT, serotonin). Previous studies have reported that C1473G polymorphism of the murine Tph2 gene leads to decreased 5-HT levels in the brain and abnormal behavioral phenotypes, such as impaired anxiety- and depression-like behaviors. In this study, to confirm the effect of the C1473G polymorphism on mouse phenotypes, we conducted a comprehensive battery of behavioral tests and measured the amounts of brain free amino acids involved in the production of 5-HT. METHODS: We obtained C57BL/6J congenic mice that were homozygous for the 1473G allele of Tph2 (1473G) and subjected them and their wild-type littermates (1473C) to a battery of behavioral tests. Using reverse-phase high-performance liquid chromatography (HPLC), we measured the amounts of free amino acids in the 5-HT and epinephrine synthetic/metabolic pathways in the frontal cortex, hippocampus, striatum, and midbrain. RESULTS: We failed to detect significant differences between genotypes in depression-like behaviors, anxiety-like behaviors, social behaviors, sensorimotor gaiting, or learning and memory, while 1473G mice exhibited a nominally significant impairment in gait analysis, which failed to reach study-wide significance. In the HPLC analysis, there were no significant differences in the amounts of 5-HT, dopamine, norepinephrine, and epinephrine in the frontal cortex, hippocampus, striatum, and midbrain. CONCLUSION: Our findings do not support the idea that congenic C57BL/6J mice carrying the 1473G allele may represent an animal model of mood disorder under normal conditions without stress.

Developmental and age-related changes to the elastic lamina of Bruch's membrane in mice.

BACKGROUND: Fibrillin-1, tropoelastin, fibulin-5, and latent transforming growth factor beta-binding protein-2 and protein-4 (LTBP-2 and LTBP-4) are essential proteins for the elastic lamina (EL). In this study, we analyzed each of these molecules in the EL of Bruch's membrane (BM) through development and aging. METHODS: C57BL/6 mice (embryonic (E) days E12.5, E15.5, and E18.5; postnatal (P) days P1, P4, and P7 and P3, P6, and P75 weeks of age) were used. To investigate localization, immunohistochemical staining (IH) was performed. Transmission electron microscopy (TEM) was used to evaluate the formation of microfibrils and tropoelastin. mRNA expression was determined by quantitative real-time PCR (qRT-PCR). RESULTS: All five proteins were expressed in the EL of BM by IH except in embryonic mice. TEM results showed that tropoelastin co-stained with microfibrils. Between 3 and 6 weeks of age, microfibrils became longer and thicker. It was difficult to evaluate the EL of BM in senile mice at 75 weeks of age because of abundant deposits which correspond to drusen. mRNA levels of each protein increased dramatically from E15.5 to P1 days and plateaued by P3 weeks as shown by qRT-PCR. CONCLUSIONS: In conclusion, these five proteins are possibly involved in elastic fiber assembly in BM. We define the date of full assembly of the EL of BM as 3 weeks of age in mice.

A new histological evaluation method to detect residual ophthalmic viscosurgical devices for cataract surgery.

PURPOSE: To establish a new evaluation method to quantify residual ophthalmic viscosurgical device (OVD) volume and corneal endothelium adhesion properties for phacoemulsification surgery. METHODS: We compared the performance of four OVDs (Viscoat®, Healon5®, Healon® and DisCoVisc®) using porcine eyes. First, OVDs were mixed with fluorescent-conjugated dextrans to render them visible under the microscope. A corneal side port was opened, followed by a continuous curvilinear capsulorhexis, and a corneal tunnel incision was made. OVDs were injected, then the lens was removed using one-handed phacoemulsification. After this procedure, the anterior segment of the eye was isolated via an equatorial incision and the tissue was immediately frozen in shimmering liquid nitrogen. Sagittal slices (20 µm) were cut with a Cryostat from limbus to limbus. Every tenth slide was imaged using a fluorescent microscope with a CCD camera. We evaluated the percentage of the corneal endothelium covered by each OVD as the OVD adhesion to corneal endothelium ratio (OAE ratio) and the volume of residual OVD in the anterior chamber. RESULTS: Viscoat® showed significantly higher endothelium coverage compared with both Healon® and DisCoVisc®. A statistically larger volume of Healon5® remained in the anterior chamber compared with Healon® and DisCoVisc®. CONCLUSION: The new evaluation methods used here provide precise quantitative analysis of OAE ratio and residual OVD volume. These results show that Viscoat® and Healon5® have a high potential for coating the corneal endothelium during phacoemulsification and aspiration surgery.

Comparison between optical coherence tomography angiography and immunolabeling for evaluation of laser-induced choroidal neovascularization.

This study aimed to investigate the differences between images obtained by optical coherence tomography angiography (OCTA) with those from immunohistochemical labeling of laser-induced choroidal neovascularization (CNV) in a mouse model. CNV was induced by laser photocoagulation (GYC-2000, NIDEK; wavelength 532 nm) in the left eyes of 10 female C57BL/6J mice aged 6 weeks. The laser parameters included a 100-µm spot, 100-ms pulse duration and 200-mW incident power to rupture Bruch's membrane. OCT and OCTA CNV images were obtained using the RS-3000 Advance (NIDEK) 5 days post-laser photocoagulation. After OCTA imaging, the isolated choroid/retinal pigment epithelium complexes were fluorescently labeled with CD31 (an endothelial cell marker), platelet-derived growth factor receptor ß (PDGFRß, a pericyte-like scaffold marker), α-smooth muscle actin (α-SMA) and collagen I. Area measurements of the lesions obtained by enface OCTA were compared with immunolabeled CD31+ CNV lesions in choroid flat-mounts. We also examined structural correlations between the PDGFRß+ pericyte-like scaffold and OCTA images. Laser-induced CNV was clearly detected by enface OCTA, appearing as a hyperflow lesion surrounded by a dark halo. Area measurements of the CNV lesion by immunolabeling were significantly larger than those obtained by enface OCTA (p = 0.006). The CNV lesion beneath the periphery of the pericyte-like scaffold was not clearly visible by enface OCTA due to the dark halo; however, the lesion was detectable as blood flow by cross-sectional OCTA and was also highly labeled by CD31. The periphery of the pericyte-like scaffold appeared to develop into subretinal fibrosis and this region was rich in myofibroblasts. Enface OCTA was unable to detect the entire area of laser-induced CNV in mice, with an undetectable portion located beneath the fibrotic periphery of the pericyte-like scaffold. Due to this OCTA fibrosis artifact, OCTA imaging has limited potential for accurately estimating CNV lesions.

AMPD1 regulates mTORC1-p70 S6 kinase axis in the control of insulin sensitivity in skeletal muscle.

BACKGROUND: Insulin resistance triggered by excess fat is a key pathogenic factor that promotes type 2 diabetes. Understanding molecular mechanisms of insulin resistance may lead to the identification of a novel therapeutic target for type 2 diabetes. AMPD1, an isoform of AMP deaminase (AMPD), is suggested to play roles in the regulation of glucose metabolism through controlling AMP-activated protein kinase (AMPK) activation. We reported that the diet-induced insulin resistance was improved in AMPD1-deficient mice compared to wild type mice. To further delineate this observation, we studied changes of insulin signaling in skeletal muscle of wild type (WT) and AMPD1-deficient mice. METHODS: Phosphorylation levels of kinases and expression levels of mTOR components were quantified by immunoblotting using protein extracts from tissues. The interaction between mTOR and Raptor was determined by immunoblotting of mTOR immunoprecipitates with anti-Raptor antibody. Gene expression was studied by quantitative PCR using RNA extracted from tissues. RESULTS: Phosphorylation levels of AMPK, Akt and p70 S6 kinase in skeletal muscle were higher in AMPD1-deficient mice compared to WT mice after high fat diet challenge, while they did not show such difference in normal chow diet. Also, no significant changes in phosphorylation levels of AMPK, Akt or p70 S6 kinase were observed in liver and white adipose tissue between WT and AMPD1-deficient mice. The expression levels of mTOR, Raptor and Rictor tended to be increased by AMPD1 deficiency compared to WT after high fat diet challenge. AMPD1 deficiency increased Raptor-bound mTOR in skeletal muscle compared to WT after high fat diet challenge. Gene expression of peroxisome proliferator-activated receptor-γ coactivator 1α and ß, downstream targets of p70 S6 kinase, in skeletal muscles was not changed significantly by AMPD1 deficiency compared to the wild type after high fat diet challenge. CONCLUSION: These data suggest that AMPD1 deficiency activates AMPK/Akt/mTORC1/p70 S6 kinase axis in skeletal muscle after high fat diet challenge, but not in normal chow diet. These changes may contribute to improve insulin resistance.

Enhanced detection of Porcine reproductive and respiratory syndrome virus in fixed tissues by in situ hybridization following tyramide signal amplification.

This study evaluated the sensitivity of biotinyl-tyramide-based in situ hybridization (TISH) method by comparison with chromogenic in situ hybridization (CISH) and immunohistochemical staining (IHC) methods. This study also determined the effect of fixative and fixation time on the detection of Porcine reproductive and respiratory syndrome virus (PRRSV) in paraffin-embedded tissues. Lung samples were fixed in 4% paraformaldehyde (PFA) or 10% neutral buffered formalin (NBF) for various times before paraffin embedding. Of 30 paraffin-embedded lung samples, fixed for 1 day in 4% PFA or 10% NBF, 18 (60%) were positive for PRRSV by nested reverse transcription polymerase chain reaction (nRT-PCR). All 18 lung samples (100%) also were positive for PRRSV by TISH, but only 10 of these 18 specimens (56%) were positive for PRRSV by IHC and CISH. We demonstrated that TISH can detect PRRSV RNA in paraffin-embedded tissues after up to 90 days of fixation. PRRSV nucleic acids and antigens were better preserved in 4% PFA than in 10% NBF. Compared with CISH and IHC testing methods, TISH appeared to be more sensitive for the detection of PRRSV in paraffin-embedded tissues.

AMPD1: a novel therapeutic target for reversing insulin resistance.

BACKGROUND: Insulin resistance is one of the hallmark manifestations of obesity and Type II diabetes and reversal of this pathogenic abnormality is an attractive target for new therapies for Type II diabetes. A recent report that metformin, a drug known to reverse insulin resistance, demonstrated in vitro the metformin can inhibit AMP deaminase (AMPD) activity. Skeletal muscle is one of the primary organs contributing to insulin resistance and that the AMPD1 gene is selectively expressed at high levels in skeletal muscle. METHODS: Recognizing the background above, we asked if genetic disruption of the AMPD1 gene might ameliorate the manifestations of insulin resistance. AMPD1 deficient homozygous mice and control mice fed normal chow diet or a high-fat diet, and blood analysis, glucose tolerance test and insulin tolerance test were performed. Also, skeletal muscle metabolism and gene expression including nucleotide levels and activation of AMP activated protein kinase (AMP kinase) were evaluated in both conditions. RESULTS: Disruption of the AMPD1 gene leads to a less severe state of insulin resistance, improved glucose tolerance and enhanced insulin clearance in mice fed a high fat diet. Given the central role of AMP kinase in insulin action, and its response to changes in AMP concentrations in the cell, we examined the skeletal muscle of the AMPD1 deficient mice and found that they have greater AMP kinase activity as evidenced by higher levels of phosphorylated AMP kinase. CONCLUSIONS: Taken together these data suggest that AMPD may be a new drug target for the reversal of insulin resistance and the treatment of Type II diabetes.

Effect of isolated AMP deaminase deficiency on skeletal muscle function.

Mutation of the AMP deaminase 1 (AMPD1) gene, the predominate AMPD gene expressed in skeletal muscle, is one of the most common inherited defects in the Caucasian population; 2-3% of individuals in this ethnic group are homozygous for defects in the AMPD1 gene. Several studies of human subjects have reported variable results with some studies suggesting this gene defect may cause symptoms of a metabolic myopathy and/or easy fatigability while others indicate individuals with this inherited defect are completely asymptomatic. Because of confounding problems in assessing muscle symptoms and performance in human subjects with different genetic backgrounds and different environmental experiences such as prior exercise conditioning and diet, a strain of inbred mice with selective disruption of the AMPD1 was developed to study the consequences of muscle AMPD deficiency in isolation. Studies reported here demonstrate that these animals are a good metabolic phenocopy of human AMPD1 deficiency but they exhibit no abnormalities in muscle performance in three different exercise protocols.

AMPD2 regulates GTP synthesis and is mutated in a potentially treatable neurodegenerative brainstem disorder.

Purine biosynthesis and metabolism, conserved in all living organisms, is essential for cellular energy homeostasis and nucleic acid synthesis. The de novo synthesis of purine precursors is under tight negative feedback regulation mediated by adenosine and guanine nucleotides. We describe a distinct early-onset neurodegenerative condition resulting from mutations in the adenosine monophosphate deaminase 2 gene (AMPD2). Patients have characteristic brain imaging features of pontocerebellar hypoplasia (PCH) due to loss of brainstem and cerebellar parenchyma. We found that AMPD2 plays an evolutionary conserved role in the maintenance of cellular guanine nucleotide pools by regulating the feedback inhibition of adenosine derivatives on de novo purine synthesis. AMPD2 deficiency results in defective GTP-dependent initiation of protein translation, which can be rescued by administration of purine precursors. These data suggest AMPD2-related PCH as a potentially treatable early-onset neurodegenerative disease.

A transgenic medaka line with visible markers for genotypic and phenotypic sex.

Accurate genotyping of sex is required for correct interpretation in any in vivo assays with endocrine disrupting chemicals (EDCs). Visible markers for genotypic sex, if reliable, simplify assays because time-consuming PCR-based genotyping can be skipped. Here, we describe a line of Japanese medaka with a brain-expressed green fluorescent protein (GFP) transgene inserted near the sex-determining locus. When used with a white pigment cell marker, genotypic sex can be determined reliably as early as 3 days after fertilization (well before gonadal sex differentiation). No recombinants were found in more than 2000 progenies. We also introduced a strong ovarian GFP marker into the line with these genetic sex markers, so that phenotypic sex can also be determined reliably at 8 days after hatching. Well-known sex reversal protocols using exogenous steroid treatments of embryos were monitored by this transgenic line, demonstrating the line to be a useful tool for in vivo studies utilizing gonadal sex differentiation of the medaka, especially for screenings of potential estrogenic and androgenic EDCs.

AMP deaminase 3 plays a critical role in remote reperfusion lung injury.

Remote reperfusion lung injury following skeletal muscle ischemia and reperfusion accounts for high morbidity and mortality. AMP deaminase (AMPD), a key enzyme for nucleotide cycle, has been implicated in the regulation of this phenomenon. However, the function of Ampd2 and Ampd3 subtype has not been elucidated in remote reperfusion rodent lung injury. We utilized AMPD3 and AMPD2-deficient mice. The two types of AMPD-deficient mice and wild-type (WT) littermates were subjected to ischemia-reperfusion injury. After 3h bilateral hind-limb ischemia and reperfusion, AMPD3 mRNA, AMPD activity and inosine monophosphate (IMP) increased significantly in WT and AMPD2-deficient mice lungs, while they did not show significant alterations in AMPD3-deficient mice lungs. Genetic inactivation of Ampd3 resulted in markedly accelerated myeloperoxidase (MPO) activity along with exaggerated neutrophils infiltration and hemorrhage in the lungs compared to WT and AMPD2-deficient mice, furthermore, IMP treatment significantly attenuated MPO activity and neutrophils infiltration in WT and the two types of AMPD-deficient mice lungs after 3h reperfusion. These findings demonstrate for the first time in AMP-deficient mice models that AMPD3 plays a critical role in remote reperfusion lung injury via generation of IMP and validate the potential to use IMP into the clinical arena to attenuate remote ischemia-reperfusion lung injury.

Synaptosomal-associated protein 25 mutation induces immaturity of the dentate granule cells of adult mice.

BACKGROUND: Synaptosomal-associated protein, 25 kDa (SNAP-25) regulates the exocytosis of neurotransmitters. Growing evidence suggests that SNAP-25 is involved in neuropsychiatric disorders, such as schizophrenia, attention-deficit/hyperactivity disorder, and epilepsy. Recently, increases in anxiety-related behaviors and epilepsy have been observed in SNAP-25 knock-in (KI) mice, which have a single amino acid substitution of Ala for Ser187. However, the molecular and cellular mechanisms underlying the abnormalities in this mutant remain unknown. RESULTS: In this study, we found that a significant number of dentate gyrus (DG) granule cells was histologically and electrophysiologically similar to immature DG neurons in the dentate gyrus of the adult mutants, a phenomenon termed the "immature DG" (iDG). SNAP-25 KI mice and other mice possessing the iDG phenotype, i.e., alpha-calcium/calmodulin-dependent protein kinase II heterozygous mice, Schnurri-2 knockout mice, and mice treated with the antidepressant fluoxetine, showed similar molecular expression patterns, with over 100 genes similarly altered. A working memory deficit was also identified in mutant mice during a spontaneous forced alternation task using a modified T-maze, a behavioral task known to be dependent on hippocampal function. Chronic treatments with the antiepileptic drug valproate abolished the iDG phenotype and the working memory deficit in mutants. CONCLUSIONS: These findings suggest that the substitution of Ala for Ser187 in SNAP-25 induces the iDG phenotype, which can also be caused by epilepsy, and led to a severe working memory deficit. In addition, the iDG phenotype in adulthood is likely an endophenotype for at least a part of some common psychiatric disorders.

Deficiency of schnurri-2, an MHC enhancer binding protein, induces mild chronic inflammation in the brain and confers molecular, neuronal, and behavioral phenotypes related to schizophrenia.

Schnurri-2 (Shn-2), an nuclear factor-κB site-binding protein, tightly binds to the enhancers of major histocompatibility complex class I genes and inflammatory cytokines, which have been shown to harbor common variant single-nucleotide polymorphisms associated with schizophrenia. Although genes related to immunity are implicated in schizophrenia, there has been no study showing that their mutation or knockout (KO) results in schizophrenia. Here, we show that Shn-2 KO mice have behavioral abnormalities that resemble those of schizophrenics. The mutant brain demonstrated multiple schizophrenia-related phenotypes, including transcriptome/proteome changes similar to those of postmortem schizophrenia patients, decreased parvalbumin and GAD67 levels, increased theta power on electroencephalograms, and a thinner cortex. Dentate gyrus granule cells failed to mature in mutants, a previously proposed endophenotype of schizophrenia. Shn-2 KO mice also exhibited mild chronic inflammation of the brain, as evidenced by increased inflammation markers (including GFAP and NADH/NADPH oxidase p22 phox), and genome-wide gene expression patterns similar to various inflammatory conditions. Chronic administration of anti-inflammatory drugs reduced hippocampal GFAP expression, and reversed deficits in working memory and nest-building behaviors in Shn-2 KO mice. These results suggest that genetically induced changes in immune system can be a predisposing factor in schizophrenia.

Comprehensive behavioral analysis of pituitary adenylate cyclase-activating polypeptide (PACAP) knockout mice.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide acting as a neurotransmitter, neuromodulator, or neurotrophic factor. PACAP is widely expressed throughout the brain and exerts its functions through the PACAP-specific receptor (PAC(1)). Recent studies reveal that genetic variants of the PACAP and PAC(1) genes are associated with mental disorders, and several behavioral abnormalities of PACAP knockout (KO) mice are reported. However, an insufficient number of backcrosses was made using PACAP KO mice on the C57BL/6J background due to their postnatal mortality. To elucidate the effects of PACAP on neuropsychiatric function, the PACAP gene was knocked out in F1 hybrid mice (C57BL/6J × 129SvEv) for appropriate control of the genetic background. The PACAP KO mice were then subjected to a behavioral test battery. PACAP deficiency had no significant effects on neurological screen. As shown previously, the mice exhibited significantly increased locomotor activity in a novel environment and abnormal anxiety-like behavior, while no obvious differences between genotypes were shown in home cage (HC) activity. In contrast to previous reports, the PACAP KO mice showed normal prepulse inhibition (PPI) and slightly decreased depression-like behavior. Previous study demonstrates that the social interaction (SI) in a resident-intruder test was decreased in PACAP KO mice. On the other hand, we showed that PACAP KO mice exhibited increased SI in Crawley's three-chamber social approach test, although PACAP KO had no significant impact on SI in a HC. PACAP KO mice also exhibited mild performance deficit in working memory in an eight-arm radial maze (RM) and the T-maze (TM), while they did not show any significant abnormalities in the left-right discrimination task in the TM. These results suggest that PACAP has an important role in the regulation of locomotor activity, social behavior, anxiety-like behavior and, potentially, working memory.

AMPD3-deficient mice exhibit increased erythrocyte ATP levels but anemia not improved due to PK deficiency.

AMP deaminase (AMPD) catalyzes AMP to IMP and plays an important role in energy charge and nucleotide metabolism. Human AMPD3 deficiency is a type of erythrocyte-specific enzyme deficiency found in individuals without clinical symptoms, although an increased level of ATP in erythrocytes has been reported. To better understand the physiological and pathological roles of AMPD3 deficiency, we established a line of AMPD3-deficient [A3(-/-)] mice. No AMPD activity and a high level of ATP were observed in erythrocytes of these mice, similar to human RBC-AMPD3 deficiency, while other characteristics were unremarkable. Next, we created AMPD3 and pyruvate kinase (PK) double-deficient [PKA(-/-,-/-)] mice by mating A3(-/-) mice with CBA-Pk-1slc/Pk-1slc mice [PK(-/-)], a spontaneous PK-deficient strain showing hemolytic anemia. In PKA(-/-,-/-) mice, the level of ATP in red blood cells was increased 1.5 times as compared to PK(-/-) mice, although hemolytic anemia in those animals was not improved. In addition, we observed osmotic fragility of erythrocytes in A3(-/-) mice under fasting conditions. In contrast, the ATP level in erythrocytes was elevated in A3(-/-) mice as compared to the control. In conclusion, AMPD3 deficiency increases the level of ATP in erythrocytes, but does not improve anemia due to PK deficiency and leads to erythrocyte dysfunction.