Gender-Dependent Effects of Maternal Immune Activation on the Behavior of Mouse Offspring

Autism spectrum disorders are neurodevelopmental disorders characterized by two core symptoms; impaired social interactions and communication, and ritualistic or repetitive behaviors. Both epidemiological and biochemical evidence suggests that a subpopulation of autistics may be linked to immune perturbations that occurred during fetal development. These findings have given rise to an animal model, called the “maternal immune activation” model, whereby the offspring from female rodents who were subjected to an immune stimulus during early or mid-pregnancy are studied. Here, C57BL/6 mouse dams were treated mid-gestation with saline, lipopolysaccharide (LPS) to mimic a bacterial infection, or polyinosinic:polycytidylic acid (Poly IC) to mimic a viral infection. Autism-associated behaviors were examined in the adult offspring of the treated dams. Behavioral tests were conducted to assess motor activity, exploration in a novel environment, sociability, and repetitive behaviors, and data analyses were carried independently on male and female mice. We observed a main treatment effect whereby male offspring from Poly IC-treated dams showed reduced motor activity. In the marble burying test of repetitive behavior, male offspring but not female offspring from both LPS and Poly IC-treated mothers showed increased marble burying. Our findings indicate that offspring from mothers subjected to immune stimulation during gestation show a gender-specific increase in stereotyped repetitive behavior.     

Acoustic Signals in Mouse Maternal Behavior: Retrieving and Cannibalism

The purpose of this study was to show that juvenile acoustic signals are not essential prerequisites for inhibiting parental aggression or for eliciting maternal care. This was demonstrated by comparing the behavior of Rb-3 and GFF+/+ female mice with normal audition and that of a deaf mutant GFF dn/dn. 243 ♀♀ and 609 litters were used to measure cannibalism and 54 ♀♀ to measure speed of retrieval. Neither rate of cannibalism nor speed of retrieval were modified by deafness, thus verifying the proposed hypothesis.     

Effects of Amphetamine on Protein Synthesis and Energy Metabolism in Mouse Brain: Role of Drug-Induced Hyperthermia

Abstract: Changes in brain protein synthesis activity, and in brain levels of glucose, glycogen, and several high-energy phosphate metabolites, were evaluated under conditions of amphetamine-induced hyperthermia in mice. Protein synthesis showed a striking dependence on rectal temperature ( T _R), falling abruptly at T _R above 40°C. A similar result was obtained following direct heating of the animals. Protein synthesis activity in liver showed the same temperature dependence observed for brain. Increased synthesis of a protein with characteristics of the major mammalian stress protein, hsp 70, was demonstrated in both brain and liver following amphetamine administration. Brain protein synthesis showed significant recovery within 2 h after amphetamine administration whereas that of liver remained below 30% of control activity, suggesting significant temporal and quantitative differences in the response of individual tissues to elevated temperatures. Brain glycogen levels after amphetamine administration were significantly lower under conditions of ambient temperature which resulted in more severe drug-induced hyperthermia but did not correlate as strikingly as protein synthesis with the temperatures of individual animals. Brain glycogen also fell in animals whose temperatures were increased by brief exposure at high ambient temperature. Brain glucose levels did not consistently change with hyperthermia. Slight decreases in high-energy phosphates with increasing T _R were likely the result of fixation artifact. These results demonstrate the fundamental role of hyperthermia in the reduction of protein synthesis in brain and other tissues by amphetamine, and suggest that temperature also constitutes a significant source of variability in the effects of this drug on brain energy metabolism, in particular glycogenolysis.     

溶酶体抑制剂对小鼠行为学与黑质多巴胺神经元的影响

目的初步探讨溶酶体抑制剂对小鼠行为学及多巴胺神经元功能的影响。方法于小鼠右侧中脑黑质（SN）区,立体定向微量注入溶酶体抑制剂和蛋白酶体抑制剂。观察阿朴吗啡诱导的小鼠旋转行为改变以及行为学变化;检测黑质区酪氨酸羟化酶（TH）阳性细胞数。结果阿朴吗啡未能诱导出小鼠旋转行为。蛋白酶体抑制剂组为10～15圈。多巴胺损害程度与溶酶体的剂量相关。磷酸氯喹25μmol/L时多巴胺神经元几乎没有损害作用;50μmol/L时局部区域多巴胺神经元有轻微损害;100μmol/L时损害明显;200μmol/L时黑质区注射局部神经元损害最严重,黑质注射区未见有神经元存在。1～4周呈现出较为明显逐渐恢复的特点。结论溶酶体功能抑制与多巴胺神经元凋亡相关,不同剂量溶酶体抑制剂在不同时间对多巴胺神经功能的损害不同。     

Distinct Effects of Different Phosphatidylglycerol Species on Mouse Keratinocyte Proliferation

We have previously shown that liposomes composed of egg-derived phosphatidylglycerol (PG), with a mixed fatty acid composition (comprising mainly palmitate and oleate), inhibit the proliferation and promote the differentiation of rapidly dividing keratinocytes, and stimulate the growth of slowly proliferating epidermal cells. To determine the species of PG most effective at modulating keratinocyte proliferation, primary mouse keratinocytes were treated with different PG species, and proliferation was measured. PG species containing polyunsaturated fatty acids were effective at inhibiting rapidly proliferating keratinocytes, whereas PG species with monounsaturated fatty acids were effective at promoting proliferation in slowly dividing cells. Thus, palmitoyl-arachidonyl-PG (16∶0/20∶4), palmitoyl-linoleoyl-PG (16∶0/18∶2), dilinoleoyl-PG (18∶2/18∶2) and soy PG (a PG mixture with a large percentage of polyunsaturated fatty acids) were particularly effective at inhibiting proliferation in rapidly dividing keratinocytes. Conversely, palmitoyl-oleoyl-PG (16∶0/18∶1) and dioleoyl-PG (18∶1/18∶1) were especially effective proproliferative PG species. This result represents the first demonstration of opposite effects of different species of a single class of phospholipid and suggests that these different PG species may signal to diverse effector enzymes to differentially affect keratinocyte proliferation and normalize keratinocyte proliferation. Thus, different PG species may be useful for treating skin diseases characterized by excessive or insufficient proliferation.     

17β-雌二醇快速诱导静止状态小鼠囊胚细胞胞内钙离子增加

用钙离子荧光探针fluo-3/AM标记囊胚细胞内钙离子,用激光共聚焦扫描显微镜连续检测17β-雌二醇(17β-E2)作用所致囊胚胞内钙离子浓度的动态变化,用荧光显微镜检测偶联牛血清白蛋白的17β-雌二醇(E2-BSA)所致囊胚胞内钙离子浓度的改变,以及在去钙镁M2液、传统雌激素受体阻断剂tamoxifen和磷脂酶C特异抑制剂U73122作用下17β-E2所致囊胚细胞内钙离子浓度的改变。结果显示:17β-E2和E2-BSA均可引起静止状态囊胚细胞内[Ca2 ]的快速升高;17β-E2诱导的囊胚细胞内[Ca2 ]的快速升高不依赖于胞外钙离子的内流,且不被传统雌激素受体阻断剂所阻断,而磷脂酶C特异性抑制剂可明显抑制该效应。     

The Vasculome of the Mouse Brain

The blood vessel is no longer viewed as passive plumbing for the brain. Increasingly, experimental and clinical findings suggest that cerebral endothelium may possess endocrine and paracrine properties – actively releasing signals into and receiving signals from the neuronal parenchyma. Hence, metabolically perturbed microvessels may contribute to central nervous system (CNS) injury and disease. Furthermore, cerebral endothelium can serve as sensors and integrators of CNS dysfunction, releasing measurable biomarkers into the circulating bloodstream. Here, we define and analyze the concept of a brain vasculome, i.e. a database of gene expression patterns in cerebral endothelium that can be linked to other databases and systems of CNS mediators and markers. Endothelial cells were purified from mouse brain, heart and kidney glomeruli. Total RNA were extracted and profiled on Affymetrix mouse 430 2.0 micro-arrays. Gene expression analysis confirmed that these brain, heart and glomerular preparations were not contaminated by brain cells (astrocytes, oligodendrocytes, or neurons), cardiomyocytes or kidney tubular cells respectively. Comparison of the vasculome between brain, heart and kidney glomeruli showed that endothelial gene expression patterns were highly organ-dependent. Analysis of the brain vasculome demonstrated that many functionally active networks were present, including cell adhesion, transporter activity, plasma membrane, leukocyte transmigration, Wnt signaling pathways and angiogenesis. Analysis of representative genome-wide-association-studies showed that genes linked with Alzheimer’s disease, Parkinson’s disease and stroke were detected in the brain vasculome. Finally, comparison of our mouse brain vasculome with representative plasma protein databases demonstrated significant overlap, suggesting that the vasculome may be an important source of circulating signals in blood. Perturbations in cerebral endothelial function may profoundly affect CNS homeostasis. Mapping and dissecting the vasculome of the brain in health and disease may provide a novel database for investigating disease mechanisms, assessing therapeutic targets and exploring new biomarkers for the CNS.     

溴氰菊酯对鼠脑蛋白质磷酸化作用的影响

以小鼠大脑碎片与[γ-~(32)P]ATP一起保温,观察到溴氰菊酯对蛋白1—3磷酸化的刺激作用和对4、5磷酸化的抑制作用,表明溴氰菊酯对大脑蛋白质磷酸化产生了影响。从鼠脑分离了C、D、S三个组分,分别进行的蛋白质磷酸化试验结果表明,C、D组分可能是重要的磷酸化部位。 蛋白1、2、3的磷酸化明显地受到溴氰菊酯的刺激,这三个蛋白质可能是“蛋白Ⅲb”的几种形式。溴氰菊酯对“蛋白Ⅲb”磷酸化的刺激,可能会影响神经末梢的神经激素释放,从而影响到与其相关的某些神经功能。     

性激素对成年小鼠脑微管蛋白合成的影响

以3H-秋水仙碱为探针,测定小鼠脑微管蛋白含量．结果表明雌性激素具有显著的促进成年鼠脑微管蛋白合成的作用．与雌性激素相比,雄性激素促进脑微管蛋白合成的作用较弱．特别值得指出的是雌性激素促进脑微管蛋白合成的作用发生在脑发育的临界期之外,而此时甲状腺激素早已丧失了促进脑微管蛋白合成的作用．因此雌性激素在维护成年脑结构和功能的完整完善方面起着重要作用,而且这种作用可能会获得新的应用．     

Effects of the Development of Thyroid and Sex Glands on the Synthesis of Brain Tubulin in Chick and Mouse Embryos

Using the specific affinity of tubulin for colchicine and the strong absorption of tubulin to DEAEion exchangers at neutral pH and moderate ionic strength,the amounts of tubulin in the brain fromboth mice and chicks during different developing stages were measured by ~3H-colchicine assay(expressed as colchicine binding activity).The results show that the rate oftubulin synthesis reacheda peak value during the critical period of brain development.This is exactly the period during whichthe organization and function of thyroid are being perfected.Besides,during breeding period,thedifference of tubulin contents between male and female is significant(P<0.001).The synthesis oftubulin is strictly sex dependent(this phenomenon appeared only during sex maturation stage).It issuggested that sexual hormones might exert their effect on tubulin synthesis.     

Regulation of Prenatal and Postnatal Protein Synthesis in Mouse Brain

Abstract: Regulation of protein synthesis during prenatal and postnatal brain development was examined using postmitochondrial supernatant (PMS) fractions and isolated ribosome-pH 5 enzyme systems from fetal, neonatal, and adult neural tissue. The rate of polyuridylic acid (poly-U)-dependent protein synthetic activity was inversely proportional to the endogenous rate of protein synthesis in either the PMS fractions or ribosomal preparations. A careful analysis of the kinetics of the poly-U-dependent polypeptide synthesis revealed that there was a lag in the time at which certain of the PMS preparations could begin to utilize the poly-U template as sole source of mRNA. The lag period was dependent upon the developmental age of the neural tissue used and the Mg²⁺ concentration of the protein synthesis reaction. Since previous work reported that the observed developmental decrease in the rate of polypeptide synthesis utilizing a poly-U template could not be measured in a purified ribosomal-pH 5 enzyme system, ribosomes were obtained by several isolation techniques to determine if the purification procedure might have affected the ribosomes in some manner by removing a specific protein(s) involved in ribosome-cytosol interactions. At 6 mM-Mg²⁺ the rate of poly-U-dependent protein synthesis was inversely proportional to the rate of endogenous synthesis and depended upon the method used to isolate the ribosomes: microsomes ∼Triton X-100-treated < DOC-treated < KCl-treated. However, there was no age-dependent effect with any of the ribosomal preparations. The data suggest that there is a developmental modulating effect of ribosomal activity in PMS preparations which is not found in association with the isolated ribosome-pH 5 enzyme protein synthesizing system.     

Distinct Brain Systems Mediate the Effects of Nociceptive Input and Self-Regulation on Pain

Cognitive self-regulation can strongly modulate pain and emotion. However, it is unclear whether self-regulation primarily influences primary nociceptive and affective processes or evaluative ones. In this study, participants engaged in self-regulation to increase or decrease pain while experiencing multiple levels of painful heat during functional magnetic resonance imaging (fMRI) imaging. Both heat intensity and self-regulation strongly influenced reported pain, but they did so via two distinct brain pathways. The effects of stimulus intensity were mediated by the neurologic pain signature (NPS), an a priori distributed brain network shown to predict physical pain with over 90% sensitivity and specificity across four studies. Self-regulation did not influence NPS responses; instead, its effects were mediated through functional connections between the nucleus accumbens and ventromedial prefrontal cortex. This pathway was unresponsive to noxious input, and has been broadly implicated in valuation, emotional appraisal, and functional outcomes in pain and other types of affective processes. These findings provide evidence that pain reports are associated with two dissociable functional systems: nociceptive/affective aspects mediated by the NPS, and evaluative/functional aspects mediated by a fronto-striatal system.     

性类固醇激素对去势和未去势雄鼠脑微管蛋白合成的影响

雌性激素在某些方面改变着神经功能。在雌性激素敏感的神经元中,雌二醇调节各种ＲＮＡ的表达,包括ｒＲＮＡ、神经多肽和神经传导物受体。这种影响可能伴有在基因产物合成方面的相应改变或翻译后修饰。最近的研究已经揭示雌性激素能改变轴突生长以及突触密度。在我们以前的实验中注意到性别对脑微管蛋白合成的影响,也发现雌性激素可刺激雄鼠脑微管蛋白合成。雌性激素可能是成年动物神经突生长的一种调节物。这种假设已经得到深入研究和证实;发现性激素对大脑皮质、海马、中脑和间脑神经元的形态和数量有着重要的影响,并改变神经突生长和突触的连接。实验分为去势（ＧＭ）和未去势（ＩＭ）两组。第一组（ＧＭ）为１月龄雄鼠,去势后１０天分为睾丸酮（ＧＭＴ）、雌二醇（ＧＭＥ）、雌二醇加孕酮（ＧＭＥＰ）三个治疗实验组和一个对照组（ＧＭＣ）。第二组（ＩＭ）为老年雄鼠,分为睾丸酮（ＩＭＴ）、雌二醇（ＩＭＥ）、雌二醇加孕酮（ＩＭＥＰ）三个治疗组和对照组（ＩＭＣ）。对照组注射芝麻油。处理结束,迅速断头,制备脑匀浆液。取上清液进行３Ｈ秋水仙碱标记反应,液闪计数,测定脑微管蛋白的生成量。实验结果如表一：微管蛋白每分钟的放射量在ＧＭ组中,ＧＭＥ比ＧＭＣ增加３７％～４３％,Ｇ     

Defined Cell Types in Superior Colliculus Make Distinct Contributions to Prey Capture Behavior in the Mouse

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Aging of Brain: Role of Estrogen

The brain undergoes many structural and functional changes during aging. Some of these changes are regulated by estrogens which act mainly through their intracellular receptors, estrogen receptor ERα and ERβ. The expression of these receptors is regulated by several factors including their own ligand estrogen, and others such as growth hormone and thyroid hormone. The levels of these factors decrease during aging which in turn influence estrogen signaling leading to alterations in brain functions. In the present paper, we review the effects of aging on brain structure and function, and estrogen action and signaling during brain aging. The findings suggest key role of estrogen in the maintenance of brain functions during aging.     

The Contribution of Citrate to the Synthesis of Acetyl Units in Synaptosomes of Developing Rat Brain

Abstract: The activities of pyruvate dehydrogenase, citrate synthase, and choline acetyltransferase in rat brain synaptosomes increased during on-togenesis by 3 and 14 times, respectively. Activity of ATP-citrate lyase decreased by 26% during the same period. Pyruvate consumption by synapto-somes from 1-day-old animals was 40% lower than that found in older rats; however, citrate efflux from intrasynaptosomal mitochondria in immature synaptosomes was over twice as high as that in mature ones. The rates of production of synaptoplasmic acetyl-CoA, ATP-citrate lyase were 1.03, 1.40, and 0.49 nmol/min/mg protein in 1-, 10-day-old, and adult rats, respectively. 3-Bromopyruvate (0.5 m M ) inhibited pyruvate consumption by 70% and caused a complete block of citrate utilization by citrate lyase in every age group. Parameters of citrate metabolism in cerebellar synaptosomes were the same as those in cerebral ones. These data indicate that production of acetyl-CoA. from citrate in synaptoplasm may be regulated either by adaptative, age-dependent changes in permeability and carrier capacity of the mitochondrial membrane or by the inhibition of synthesis of intramitochondrial acetyl-CoA. ATP-citrate lyase activity is not a rate-limiting factor in this process. Metabolic fluxes of pyruvate to cytoplasmic citrate and acetyl-CoA. are presumably the same in both cholinergic and noncholinergic nerve endings. The significance of citrate release from intrasynaptosomal mitochondria as a regulatory step in acetylcholine synthesis is discussed.     

Distinct, Developmentally Regulated Brain mRNAs Direct the Synthesis of Neurotransmitter Transporters

The Xenopus laevis oocyte expression system was utilized to define developmental and structural properties of neurotransmitter transporter mRNAs and the pharmacological characteristics of encoded carriers independent of the complexities of brain tissue preparations. Poly(A)+ RNA from dissected brain regions of neonatal and adult rats was microinjected into Xenopus oocytes and the expression of Na(+)-dependent neurotransmitter transporters determined 48 h later. Transport studies conducted with oocytes injected with RNAs derived from juvenile rat tissues indicate a region- and transporter-specific, postnatal increase in mRNA abundance as a major factor in the developmental changes observed for brain high-affinity amino acid uptake systems. Both L-glutamic acid (Glu) and gamma-aminobutyric acid (GABA) uptake systems were detectable by day 3 in postnatal forebrain mRNA and became progressively enriched during the next 2 weeks of forebrain development. In contrast, brainstem Glu and GABA transporter enrichment was 60-70% of adult values by day 3 and exceeded adult levels by day 10. Parallel determinations of L-glutamic acid decarboxylase mRNA abundance during development argue for distinct regulatory influences on mRNAs directing transmitter synthesis and reuptake. Glycine uptake could not be detected at any point of forebrain development and exhibited a gradual postnatal rise to adult levels over the first 3 postnatal weeks of brainstem development. Uptake studies conducted with well-characterized inhibitors of Glu, GABA, dopamine, and choline transport (D-aspartate, nipecotic acid, nomifensine, and hemicholinium-3, respectively) revealed that oocyte transporters encoded by adult rat brain mRNAs retained antagonist sensitivities exhibited by in vitro brain preparations. In addition, a differential regional sensitivity to the Glu transport antagonist dihydrokainate (1 mM) was observed, lending support to previous reports of region-specific Glu transporter subtypes. To determine the structural diversity present among brain transporter mRNAs, poly(A)+ RNA was size-fractionated on linear (10-31%) sucrose density gradients prior to oocyte injection. These experiments revealed two mRNA size classes (2.4-3.0 kb, 4.0-4.5 kb) independently capable of directing the synthesis of Glu, GABA, and glycine transporters. In regions other than the cerebellum, Glu and GABA transporter activities migrated as single, yet distinct, peaks of 4.0-4.5 kb. In contrast, both Glu and GABA transporters exhibited major peaks of activity at 2.5-3.0 kb with size-fractionated cerebellar mRNA. Brainstem glycine uptake exhibited a broad sedimentation profile, with peaks apparent at 2.4 and 4.0 kb. Taken together, these findings indicate previously unappreciated complexity in mRNA structure and regulation which underlies the expression of amino acid neurotransmitter uptake systems in the rodent CNS.     

Rapid Preparation of a Distinct Lysosomal Population from Myelinating Mouse Brain Using Percoll Gradients

To study the vesicular lysosome-associated transport and the metabolism of some brain macromolecules (in particular, sialoglycoconjugates), we developed a rapid procedure to obtain a distinct lysosomal population starting from myelinating mouse brain. This procedure is based on an initial differential centrifugation step producing a 1,000-17,500-g fraction (P2), followed by isopycnic centrifugation of fraction P2 on a self-generated colloidal silica gel (Percoll) gradient. The heaviest subfraction thus obtained is very rich in acid hydrolase activities like beta-galactosidase, arylsulfatase A, and acid phosphatase. The enrichment of these enzymes is approximately 100-fold as compared with the starting homogenate, whereas the markers of other subcellular organelles, such as mitochondria, plasma membranes, or the Golgi apparatus, are virtually absent. The lysosomal preparation contains approximately 12-14% of the total acid hydrolase activities, with a protein yield of approximately 0.12%. Electron microscopy shows that the lysosomal fraction is composed of an approximately 90% pure population of lysosomes. Therefore, the procedure described here is suitable for obtaining a highly purified lysosome preparation from myelinating mouse brain.