Prenatal Morphine Exposure Differentially Alters Addictive and Emotional Behavior in Adolescent and Adult Rats in a Sex-Specific Manner

The effects of prenatal opioid exposure in adult animals has been widely studied, but little is known about the effects of prenatal opioid on adolescents. Most of the risk behaviors associated with drug abuse are initiated during adolescence. The developmental state of the adolescent brain makes it vulnerable to initiate drug use and susceptible to drug-induced brain changes. In this study, pregnant rats were subcutaneously injected with an increasing dose of morphine (5 mg/kg, 7 mg/kg, 10 mg/kg) for 9 days since the gestation day 11. The effects of prenatal morphine (PNM) on learning and memory, anxiety- and depressive- like behavior, morphine induced conditioned place preference (CPP) as well as locomotor sensitization were tested in both adolescent and adult rats. The results showed that: (1) PNM decreased anxiety-like behavior in both adolescent and adult female rats, but not males; (2) PNM decreased depressive-like behavior in adolescent but increased depressive -like behavior in adult females; (3) PNM increased low dose morphine induced locomotor sensitization in females; (4) PNM decreased tyrosine hydroxylase (TH) expression in the prefrontal cortex but decreased dopamine D1 receptor expression in the nucleus-accumbens (NAc) in female rats. These results suggested that PNM altered the emotional and addictive behavior mainly in female rats, with female rats being less anxiety and depressive during adolescence, but more depressive in adult, and more sensitive to low dose morphine induced locomotor activity sensitization, which might be mediated in part by the differential expression of the TH, dopamine D1 receptors in the female brain.

     

Improved Methodology for Sensitive and Rapid Quantitative Proteomic Analysis of Adult‐Derived Mouse Microglia: Application to a Novel In Vitro Mouse Microglial Cell Model

Microglia, as the resident brain immune cells, can exhibit a broad range of activation phenotypes, which have been implicated in a multitude of central nervous system disorders. Current widely studied microglial cell lines are mainly derived from neonatal rodent brain that can limit their relevance to homeostatic function and disease‐related neuroimmune responses in the adult brain. Recently, an adult mouse brain‐derived microglial cell line has been established; however, a comprehensive proteome dataset remains lacking. Here, an optimization method for sensitive and rapid quantitative proteomic analysis of microglia is described that involves suspension trapping (S‐Trap) for efficient and reproducible protein extraction from a limited number of microglial cells expected from an adult mouse brain (≈300 000). Using a 2‐h gradient on a 75‐cm UPLC column with a modified data dependent acquisition method on a hybrid quadrupole‐Orbitrap mass spectrometer, 4855 total proteins have been identified where 4698 of which are quantifiable by label‐free quantitation with a median and average coefficient of variation (CV) of 6.7% and 10.6%, respectively. This dataset highlights the high depth of proteome coverage and related quantitation precision of the adult‐derived microglial proteome including proteins associated with several key pathways related to immune response. Data are available via ProteomeXchange with identifier PXD012006.     

Posthatch Oral Estrogen Exposure Impairs Adult Reproductive Performance of Zebra Finch in a Sex-Specific Manner

We determined whether short-term, posthatch oral exposure to estradiol benzoate (EB) or the industrial surfactant octylphenol (OP) could impair the reproductive performance of zebra finches. If so, naturally occurring phytoestrogens and xenoestrogens might influence reproduction in wild populations. Chicks were given oral administration of 10 or 100 nmol EB per gram of body mass (earlier work showed the latter to be the minimum oral dose required to maximally masculinize female song nuclei) or an equimolar amount of OP daily from 5 through 11 days of age. Canola oil was used as a vehicle and control. Reproductive testing was done either in individual pair cages or in communal cages that permitted self-selection of mates, N = 10 pairs per group. Pairs consisted of EB-treated males and females, EB-treated males paired with canola-treated females, vice versa, and canola-treated males and females. Posthatch EB treatment produced sex-specific impairments in reproduction that, in some instances, were additive when both sexes were treated. Egg production was reduced and egg breakage was increased in 100 nmol/g EB-treated male and female pairs. The incidence of missing eggs was increased in 10 nmol/g EB-treated male and female pairs. Candled fertility was reduced in both groups containing 100 nmol/g EB-treated males. The number of hatched chicks was severely reduced in all EB-treated groups. No adverse effects of OP treatment were detected. These significant treatment effects (all P < 0.05) show that posthatch EB treatment profoundly disrupts the reproductive performance of zebra finches, suggesting that exposure to estrogens in the wild could impair the reproductive performance of wild populations.     

Expression of CXCL4 in microglia in vitro and in vivo and its possible signaling through CXCR3

Signaling through chemokine receptor CXCR3 in the brain has been implicated in various brain diseases, as CXCR3 and its ligands are found under these conditions. Recently, a new chemokine ligand for CXCR3 was reported. In humans, an alternatively spliced variant of CXCR3 expressed on microvascular endothelial cells, named CXCR3b, was shown to bind CXCL4. In the periphery, the cellular expression and functions of CXCL4 are well described but in the brain its expression and function are unknown. Here, we show that brain microglia are a cellular source of CXCL4 in vitro and in vivo under neurodegenerating conditions. Microglial migration induced by CXCL4 is absent in CXCR3-deficient microglia, indicating a role of CXCR3. CXCL4 furthermore attenuates lipopolysaccharide-induced microglial phagocytosis and nitric oxide production in microglia and BV-2 cells. Based on these findings, it is proposed that locally released CXCL4 may control microglia responses.     

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)¹. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)². Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers³. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation⁴ and pathologies such as inflammation⁵. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages⁶ and microglia⁷. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer''s disease and brain tumors.     

Sex Differences in Infant Rhesus Macaque Separation–Rejection Vocalizations and Effects of Prenatal Androgens

Infant and juvenile rhesus macaques exhibit many sexually dimorphic behaviors, including rough and tumble play, mounting, and time spent with nonmother females. This study investigated sex differences in infant rhesus monkey separation–rejection vocalizations (SRVs), and the effects of altering the prenatal hormone environment on these differences. Pregnant females received exogenous androgen (testosterone enanthate), an androgen antagonist (flutamide), or vehicle injections for 30 or 35 days during the second (early) or third (late) trimester of pregnancy. Control females used a greater percentage of coos and arched screams than did control males. In contrast, males used a greater percentage of geckers and noisy screams than did females. Females also had longer SRV bouts, used more calls, and used more types of vocalizations than did males. Mothers were more likely to respond to the SRVs of male infants than to the SRVs of female infants. Prenatal flutamide treatment early in gestation reduced the likelihood that mothers would respond to their male offspring, but prenatal androgen treatment had no effect on response rates of mothers to female offspring. Early, but not late, androgen treatment produced females who vocalized in a male-typical manner. Similarly, early flutamide treatment produced males who displayed more female-typical SRVs. Late flutamide treatments of females produced as much masculinization of SRVs as did early androgen treatment in females. These results demonstrate sex differences in highly emotional vocalizations in infant rhesus macaques and provide evidence that the timing and form of prenatal hormonal exposure influence such vocalizations.     

Diet,Genetics, and the Gut Microbiome Drive Dynamic Changes in Plasma Metabolites

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Relationship between microglial activation and dopaminergic neuronal loss in the substantia nigra: a time course study in a 6-hydroxydopamine model of Parkinson's disease

Cellular interactions between activated microglia and degenerating neurons in in vivo models of Parkinson's disease are not well defined. This time course study assesses the dynamics of morphological and immunophenotypic properties of activated microglia in a 6-hydroxydopamine (6-OHDA) model of Parkinson's disease. Neurodegeneration in the substantia nigra pars compacta (SNc) was induced by unilateral injection of 6-OHDA into the medial forebrain bundle. Activated microglia, identified using monoclonal antibodies: clone of antibody that detects major histocompatibility complex (MHC) class II antigens (OX6) for MHC class II, clone of antibody that detects cell surface antigen-cluster of differentiation 11b – anti-complement receptor 3, a marker for complement receptor 3 and CD 68 for phagocytic activity. Activation of microglia in the lesioned SNc was rapid with cells possessing amoeboid or ramified morphology appeared on day 1, whilst antibody clone that detects macrophage-myeloid associated antigen immunoreactivity was observed at day 3 post-lesion when there was no apparent loss of tyrosine hydroxylase (TH)+ve dopaminergic (DA) SNc neurons. Thereafter, OX6 and antibody clone that detects macrophage-myeloid associated antigen activated microglia selectively adhered to degenerating axons, dendrites and apoptotic (caspase 3+ve) DA neurons in the SNc were observed at day 7. This was followed by progressive loss of TH+ve SNc neurons, with the peak of TH+ve cell loss (51%) being observed at day 9. This study suggests that activation of microglia precedes DA neuronal cell loss and neurons undergoing degeneration may be phagocytosed prematurely by phagocytic microglia.     

大鼠脊髓小胶质细胞P2X4的表达和糖尿病病理性神经痛大鼠炎症反应和疼痛阈值的关系

摘要 目的：探究大鼠脊髓小胶质细胞P2X4的表达和糖尿病病理性神经痛大鼠炎症反应和疼痛阈值的关系。方法：通过高脂饮食结合链脲佐菌素注射诱导糖尿病病理性神经痛大鼠模型并分为3组：对照组（正常大鼠,腹腔注射载体柠檬酸盐缓冲液0.25 mL/kg）,模型组（糖尿病病理性疼痛模型,同上注射,n=15）和抑制剂组（大鼠糖尿病病理性模型,过鞘内导管注射米诺环素）,共28 d。通过MWT评估对机械刺激的手掌反应。通过双极针电极检测实验大鼠的运动神经传导速速。通过蛋白印迹分析P2X4和BDNF蛋白表达。通过RT-PCR分析炎症因子IL-1β、TNF-α和NLRP3的mRNA表达。通过蛋白印迹分析p38MAPK和p-p38MAPK的蛋白表达。结果：第2week、4week和6week,模型组MWT较对照组降低（P<0.05）,抑制剂组MWT较模型组升高（P<0.05）。第2 week,个实验组大鼠MNCV比较无差异（P>0.05）,第4week和第6week,模型组MNCV较对照组降低（P<0.05）,抑制剂组MNCV较模型组升高（P<0.05）。模型组P2X4和BDNF蛋白表达较对照组升高（P<0.05）,抑制剂组P2X4和BDNF蛋白表达较模型组降低（P<0.05）,模型组P2X4和BDNF mRNA表达较对照组升高（P<0.05）,抑制剂组P2X4和BDNF mRNA表达较模型组降低（P<0.05）。模型组IL-1β、TNF-α和NLRP3的mRNA表达较对照组升高（P<0.05）,抑制剂组IL-1β、TNF-α和NLRP3的mRNA表达较抑制剂组降低（P<0.05）。模型组p-p38MAPK蛋白表达较对照组升高（P<0.05）,抑制剂组p-p38MAPK蛋白表达较模型组降低（P<0.05）,各实验组大鼠p38MAPK蛋白表达无差异（P>0.05）。结论：大鼠脊髓小胶质细胞P2X4-BDNF信号在DNP中起重要作用,并且P2X4在DNP期间激活的脊髓小胶质细胞中表达升高,抑制小胶质细胞激活能显著降低P2X4表达和炎症水平,可防止热痛觉过敏并增加大鼠疼痛阈值。     

Sex-Specific Recruitment and Brood Sex Ratios of Eurasian Kestrels in a Seasonally and Annually Fluctuating Northern Environment

Timing of birth and food availability may select for biased offspring sex ratios when they differentially affect the reproductive value of male and female young. Here we show that early hatching date enhances more the probability of male Eurasian kestrels (Falco tinnunculus) to breed as one-year-old than that of females in a Finnish population. This rarely documented phenomenon has been previously observed in a kestrel population in the Netherlands. As kestrels in the Finnish population are migratory, our results refute the hypothesis that early-fledged males would have an advantage for early breeding only in resident populations. Contrary to the predictions, the Finnish population showed no change in brood sex ratio during the breeding season in a long-term data from 8years. As far as we know, this is the first demonstration that biased sex allocation may not occur even when it would appear to be adaptive. This result is different from the Dutch kestrel population, in which the season began with a bias towards males and ended with a bias in favour of females. We suggest that high inter-annual variation in food abundance in Finland might reduce selection for a sex ratio trend.     

Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury.     

Influences of Relatedness,Food Deprivation,and Sex on Adult Behaviors in the Group‐living Insect Forficula auricularia

The evolution of group living is generally associated with the emergence of social behaviors that ensure fitness benefits to group members. However, the expression of these behaviors may depend on group composition, which can vary over time with respect to sex, starvation status, and relatedness. Here, we investigated (1) whether adults of the group‐living European earwig, Forficula auricularia, show cooperative behaviors toward conspecifics and (2) whether sex, food availability, and relatedness shape the nature and frequency of these behaviors. We conducted a full‐factorial experiment using 108 unisexual pairs of adults, in which we manipulated these three factors and video‐recorded the earwig behaviors for 45 min. Our results revealed that adults mostly expressed self‐directed and aggressive behaviors. Nevertheless, they also showed allogrooming, a social behavior that offers scope for cooperation. Pairs of males displayed longer bouts of aggression and allogrooming (when it occurred) than pairs of females. Food deprivation had no effect on male behaviors, but females spent less time self‐grooming and walking when they were food deprived. Finally, low relatedness between adults did not influence any of the measured behaviors, but exacerbated frass production, possibly due to social stress. Overall, these results indicate the limited role of cooperation among F. auricularia adults during their group‐living phase.     

Novel role of aquaporin-4 in CD4+ CD25+ T regulatory cell development and severity of Parkinson's disease

Aquaporin-4 (AQP4) is highly expressed in mammalian brains and is involved in the pathophysiology of cerebral disorders, including stroke, tumors, infections, hydrocephalus, epilepsy, and traumatic brain injury. We found that AQP4-deficient mice were hypersensitive to stimulations such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or lipopolysaccharide compared to wild-type (WT) littermates. In a mouse model of MPTP-induced Parkinson's disease (PD), AQP4-deficient animals show more robust microglial inflammatory responses and more severe loss of dopaminergic neurons (DNs) compared with WT mice. However, a few studies have investigated the association of abnormal AQP4 levels with immune dysfunction. Here, for the first time, we report AQP4 expression in mouse thymus, spleen, and lymph nodes. Furthermore, the significantly lower numbers of CD4(+) CD25(+) regulatory T cells in AQP4-deficient mice compared to WT mice, perhaps resulting from impaired thymic generation, may be responsible for the uncontrolled microglial inflammatory responses and subsequent severe loss of DNs in the substantia nigra pars compacta in the MPTP-induced PD model. These novel findings suggest that AQP4 deficiency may disrupt immunosuppressive regulators, resulting in hyperactive immune responses and potentially contributing to the increased severity of PD or other immune-associated diseases.     

Candidate diagnostic biomarkers for neurodevelopmental disorders in children and adolescents: a systematic review

Neurodevelopmental disorders – including attention-deficit/hyperactivity disorder (ADHD), autism spectrum disorder, communication disorders, intellectual disability, motor disorders, specific learning disorders, and tic disorders – manifest themselves early in development. Valid, reliable and broadly usable biomarkers supporting a timely diagnosis of these disorders would be highly relevant from a clinical and public health standpoint. We conducted the first systematic review of studies on candidate diagnostic biomarkers for these disorders in children and adolescents. We searched Medline and Embase + Embase Classic with terms relating to biomarkers until April 6, 2022, and conducted additional targeted searches for genome-wide association studies (GWAS) and neuroimaging or neurophysiological studies carried out by international consortia. We considered a candidate biomarker as promising if it was reported in at least two independent studies providing evidence of sensitivity and specificity of at least 80%. After screening 10,625 references, we retained 780 studies (374 biochemical, 203 neuroimaging, 133 neurophysiological and 65 neuropsychological studies, and five GWAS), including a total of approximately 120,000 cases and 176,000 controls. While the majority of the studies focused simply on associations, we could not find any biomarker for which there was evidence – from two or more studies from independent research groups, with results going into the same direction – of specificity and sensitivity of at least 80%. Other important metrics to assess the validity of a candidate biomarker, such as positive predictive value and negative predictive value, were infrequently reported. Limitations of the currently available studies include mostly small sample size, heterogeneous approaches and candidate biomarker targets, undue focus on single instead of joint biomarker signatures, and incomplete accounting for potential confounding factors. Future multivariable and multi-level approaches may be best suited to find valid candidate biomarkers, which will then need to be validated in external, independent samples and then, importantly, tested in terms of feasibility and cost-effectiveness, before they can be implemented in daily clinical practice.