Trophic action of leptin on hypothalamic neurons that regulate feeding

In adult mammals, the adipocyte-derived hormone leptin acts on the brain to reduce food intake by regulating the activity of neurons in the arcuate nucleus of the hypothalamus (ARH). Here, we report that neural projection pathways from the ARH are permanently disrupted in leptin-deficient (Lepob/Lepob) mice and leptin treatment in adulthood does not reverse these neuroanatomical defects. However, treatment of Lepob/Lepob neonates with exogenous leptin rescues the development of ARH projections, and leptin promotes neurite outgrowth from ARH neurons in vitro. These results suggest that leptin plays a neurotrophic role during the development of the hypothalamus and that this activity is restricted to a neonatal critical period that precedes leptin's acute regulation of food intake in adults.     

Rapid rewiring of arcuate nucleus feeding circuits by leptin

The fat-derived hormone leptin regulates energy balance in part by modulating the activity of neuropeptide Y and proopiomelanocortin neurons in the hypothalamic arcuate nucleus. To study the intrinsic activity of these neurons and their responses to leptin, we generated mice that express distinct green fluorescent proteins in these two neuronal types. Leptin-deficient (ob/ob) mice differed from wild-type mice in the numbers of excitatory and inhibitory synapses and postsynaptic currents onto neuropeptide Y and proopiomelanocortin neurons. When leptin was delivered systemically to ob/ob mice, the synaptic density rapidly normalized, an effect detectable within 6 hours, several hours before leptin's effect on food intake. These data suggest that leptin-mediated plasticity in the ob/ob hypothalamus may underlie some of the hormone's behavioral effects.     

大鼠品系PGR-iCre构建

采用CRISPR/Cas9基因编辑技术首次制备用于子宫基因敲除的PGR-iCre大鼠。PGR-iCre大鼠与含有Flox报告基因Tdtomato大鼠(Tdtomatof/f)杂交得到PGRCre+/--Tdtomatof/+大鼠。利用real-time PCR、免疫组化、荧光检测等技术检测Tdtomato基因在PGRCre+/--Tdtomatof/+大鼠不同器官表达,间接检测PGR驱动的Cre重组酶组织特异性表达。结果表明,PGR驱动Cre重组酶在大鼠子宫、卵巢、输卵管、乳腺、下丘脑、垂体、脾脏、肾脏、肺等器官不同程度表达;未检测到Cre重组酶在心脏、肝脏、肌肉中表达。综上,PGR-iCre大鼠可作为子宫基因敲除工具鼠研究大鼠子宫表达基因的相关功能。     

Transplanted hypothalamic neurons restore leptin signaling and ameliorate obesity in db/db mice

Evolutionarily old and conserved homeostatic systems in the brain, including the hypothalamus, are organized into nuclear structures of heterogeneous and diverse neuron populations. To investigate whether such circuits can be functionally reconstituted by synaptic integration of similarly diverse populations of neurons, we generated physically chimeric hypothalami by microtransplanting small numbers of embryonic enhanced green fluorescent protein-expressing, leptin-responsive hypothalamic cells into hypothalami of postnatal leptin receptor-deficient (db/db) mice that develop morbid obesity. Donor neurons differentiated and integrated as four distinct hypothalamic neuron subtypes, formed functional excitatory and inhibitory synapses, partially restored leptin responsiveness, and ameliorated hyperglycemia and obesity in db/db mice. These experiments serve as a proof of concept that transplanted neurons can functionally reconstitute complex neuronal circuitry in the mammalian brain.     

Leptin长型受体与神经肽Y在猪前脑内共存

用原位杂交与免疫组织化学相结合的方法研究了 5头三元杂交仔猪前脑内leptin长型受体mRNA与神经肽Y(NPY)免疫反应阳性物质的共存关系。实验结果表明 ,leptin长型受体mRNA和NPY免疫反应阳性神经元分布于下丘脑漏斗核和前室周核、海马、杏仁核及大脑皮质。上述结构内一些表达leptin长型受体mRNA的神经元也含有NPY免疫反应阳性物质 ,即leptin长型受体与NPY共存于同一神经元内。提示leptin可能通过下丘脑漏斗核和室周核以及边缘系统的海马和杏仁核 ,甚至大脑皮质中表达leptin长型受体mRNA的NPY免疫反应阳性神经元调节动物的摄食、能量代谢、生长和繁殖等活动。     

Transneuronal transfer of herpes virus from peripheral nerves to cortex and brainstem

The transneuronal transfer of neurotropic viruses may represent an effective tool for tracing chains of connected neurons because replication of virus in the recipient neurons after transfer amplifies the "tracer signal." Herpes simplex virus type 1 was transferred transneuronally from forelimb and hindlimb nerves of rats to the cortical and brainstem neurons that project to the spinal enlargements to which the nerves receiving injections are connected. This transneuronal transfer of herpes simplex virus type 1 from peripheral nerves has the potential to be used to identify neurons in the brain that are related transsynaptically to different nerves and muscles.     

Hypothalamic mTOR signaling regulates food intake

The mammalian Target of Rapamycin (mTOR) protein is a serine-threonine kinase that regulates cell-cycle progression and growth by sensing changes in energy status. We demonstrated that mTOR signaling plays a role in the brain mechanisms that respond to nutrient availability, regulating energy balance. In the rat, mTOR signaling is controlled by energy status in specific regions of the hypothalamus and colocalizes with neuropeptide Y and proopiomelanocortin neurons in the arcuate nucleus. Central administration of leucine increases hypothalamic mTOR signaling and decreases food intake and body weight. The hormone leptin increases hypothalamic mTOR activity, and the inhibition of mTOR signaling blunts leptin's anorectic effect. Thus, mTOR is a cellular fuel sensor whose hypothalamic activity is directly tied to the regulation of energy intake.     

运动对高脂饮食大鼠下丘脑SOCS-3和BDNF以及瘦素抵抗的影响

目的：观察运动对高脂饮食大鼠血清和下丘脑瘦素（Leptin）以及下丘脑 瘦素长型受体（OB-Rb） 、促黑皮素受体4（MC4R）、细胞因子信号转导抑制因子-3（SOCS-3） mRNA表达和脑源性神经营养因子（ BDNF）的影响,探讨适宜运动对高脂饮食大鼠预防肥胖的部分中枢机制。方法：雄性SD鼠24只,随机分为 正常对照组（C组）,高脂模型组（H组）和高脂 60min 运动组（HE组）,每组8只。高脂模型组和高脂 60min 运动组大鼠喂饲高脂饲料,高脂饮食运动组大鼠进行60min的无负重游泳运动。持续10周。结果： 10周运动后,高脂模型组大鼠较正常对照组大鼠体重、Lee''S指数、体脂、脂体比均显著增高(p<0.05), 血清瘦素水平显著升高(p<0.05),下丘脑内OB-Rb、MC4R mRNA表达量显著降低(p<0.05),SOCS-3 mRNA表 达量显著升高(p<0.05),下丘脑BDNF含量显著降低(p<0.05);高脂60min运动组较高脂模型组大鼠体重显 著降低(p<0.01),Lee''S指数、体脂、脂体比均显著降低(p<0.05),血清瘦素水平显著升高(p<0.05),下 丘脑OB-Rb、MC4R、leptin mRNA表达量显著升高(p<0.05),下丘脑SOCS-3 mRNA表达量显著降低(p<0.05) ,下丘脑BDNF含量显著升高(p<0.05)。结论：长期适宜的运动可通过降低下丘脑SOCS-3的表达,增加下丘 脑Leptin 与受体OB-Rb的结合,降低了瘦素抵抗,激活下丘脑含 MC4R 受体的神经元,从而刺激 BDNF 的 释放,从而起到预防高脂饮食引起肥胖的作用。     

Immunohistochemistry of adenosine deaminase: implications for adenosine neurotransmission

Immunohistochemical analysis of adenosine deaminase in rat brain revealed an extensive plexus of adenosine deaminase-containing neurons in the basal hypothalamus. These neurons converged on and were most numerous in three major centers, namely, the tuberal, caudal, and postmammillary caudal magnocellular nuclei. Most other brain regions were devoid of cells containing adenosine deaminase. Some adenosine deaminase-containing neurons were retrogradely labeled with the fluorescent dye fast blue when the dye was injected into the frontal cortex and striatum. Specific populations of neurons having high levels of adenosine deaminase may release adenosine as a neurotransmitter.     

The gut and energy balance: visceral allies in the obesity wars

In addition to digesting and assimilating nutrients, the intestine and associated visceral organs play a key sensing and signaling role in the physiology of energy homeostasis. The gut, the pancreatic islets of Langerhans, elements in the portal vasculature, and even visceral adipose tissue communicate with the controllers of energy balance in the brain by means of neural and endocrine pathways. Signals reflecting energy stores, recent nutritional state, and other parameters are integrated in the central nervous system, particularly in the hypothalamus, to coordinate energy intake and expenditure. Our understanding of regulatory neural circuits and the signaling molecules that influence them has progressed rapidly, particularly after the discovery of the adipocyte hormone leptin. These discoveries have led to exploration of novel routes for obesity control, some of which involve gut-derived pathways.     

Leptin regulates striatal regions and human eating behavior

Studies of the fat-derived hormone leptin have provided key insights into the molecular and neural components of feeding behavior and body weight regulation. An important challenge lies in understanding how the rewarding properties of food interact with, and can override, physiological satiety signals and promote overeating. We used functional magnetic resonance imaging to measure brain responses in two human patients with congenital leptin deficiency who were shown images of food before and after 7 days of leptin replacement therapy. Leptin was found to modulate neural activation in key striatal regions, suggesting that the hormone acts on neural circuits governing food intake to diminish the perception of food reward while enhancing the response to satiety signals generated during food consumption.     

Construction of chimeric viruses based on pepper mild mottle virus using a modified Cre/loxP system

Cre/loxP, a site-specific recombination system, has been widely used for various purposes, including chromosomal translocations, generation of marker-free transgenic plants, tissue-specific activation of a reporter gene and efficient heterologous gene expression in plants. However, stable or transient expression of Cre recombinase in plants can cause chlorosis or necrosis. Here, we describe a modified Cre/loxP recombination system using a DNA fragment flanked with loxP sites in the same orientation in which necrosis induced by Cre recombinase in Nicotiana benthamiana leaves was alleviated. The modified system was successfully used to create functional GFP-tagged pepper mild mottle virus (PMMoV) and a chimeric virus with coat protein (CP) substitution assembled from separate pro-vector modules. Our results provide a new strategy and flexible technique to construct chimeric virus and infectious clones for plant viruses with large genomes.     

Mosaic organization of neural stem cells in the adult brain

The in vivo potential of neural stem cells in the postnatal mouse brain is not known, but because they produce many different types of neurons, they must be either very versatile or very diverse. By specifically targeting stem cells and following their progeny in vivo, we showed that postnatal stem cells in different regions produce different types of neurons, even when heterotopically grafted or grown in culture. This suggests that rather than being plastic and homogeneous, neural stem cells are a restricted and diverse population of progenitors.     

A method for estimating the number of hidden neurons in feed-forward neural networks based on information entropy

The number of hidden neurons of feed-forward neural networks is generally decided on the basis of experience. The method usually results in the lack or redundancy of hidden neurons, and causes the shortage of capacity for storing information or learning overmuch. This research proposes a new method for optimizing the number of hidden neurons based on information entropy. Firstly, an initial neural network with enough hidden neurons should be trained by a set of training samples. Secondly, the activation values of hidden neurons should be calculated by inputting the training samples that can be identified correctly by the trained neural network. Thirdly, all kinds of partitions should be tried and its information gain should be calculated, and then a decision tree for correctly dividing the whole sample space can be constructed. Finally, the important and related hidden neurons that are included in the tree can be found by searching the whole tree, and other redundant hidden neurons can be deleted. Thus, the number of hidden neurons can be decided. Taking a neural network with the best number of hidden units for tea quality evaluation as an example, the result shows that the method is effective.     

SIF干预肥胖大鼠下丘脑瘦素介导JAK/STAT信号通路的研究

通过观察SIF对肥胖大鼠下丘脑中瘦素介导的信号通路蛋白表达的影响,探讨SIF对肥胖大鼠的干预机制。采用高脂饲料饲喂大鼠,建立肥胖动物模型,并将肥胖大鼠分为4组,分别灌胃SIF,剂量为：对照组（Ⅰ组）0 mg·kg–1、低剂量组（Ⅱ组）50 mg·kg–1、中剂量组（Ⅲ组）150 mg·kg–1、高剂量组（Ⅳ组）450 mg·kg–1,并设置正常大鼠基础组（Ⅴ组）,饲喂基础饲料,并灌胃给予SIF处理等体积的羧甲基纤维素钠,连续4周。采用HE染色法观察下丘脑组织学结构变化,免疫组织化学SABC法检测下丘脑中瘦素介导的JAK/STAT信号转导通路蛋白OB Rb（瘦素受体长型）,JAK2（内源性酪氨酸蛋白激酶2）,p STAT3（磷酸化的信号转导与转录激活子3）,SOCS3（细胞因子信号3抑制因子）,NPY（神经肽Y）的表达水平。结果显示：高剂量组和基础组OB Rb,JAK2,p STAT3的阳性表达水平显著高于其他组（P＜0.05）,且前述蛋白均有随SIF的剂量增加而表达增多趋势。高剂量组和基础组的SOCS3和NPY表达水平显著较其他组低（P＜0.05）,但NPY在背内侧核和室旁核的表达随SIF的剂量增加而增多。OB Rb,JAK2,p STAT3,SOCS3和NPY均在下丘脑广泛表达,并随SIF的剂量增加而变化,提示SIF对瘦素介导的信号通路有一定作用并与机体能量的调节密切相关;SIF通过干预大鼠下丘脑瘦素JAK/STAT信号通路具有减重作用,并呈剂量依赖性。     

Selective D2 receptor actions on the functional circuitry of working memory

Prefrontal neurons engaged by working memory tasks express a sequence of phasic and tonic activations linked to a train of sensory, mnemonic, and response-related events. Here, we report that the dopamine D2 receptor selectively modulates the neural activities associated with memory-guided saccades in oculomotor delayed-response tasks yet has little or no effect on the persistent mnemonic-related activity, which is instead modulated by D1 receptors. This associates the D2 receptor with a specific component of working memory circuitry and fractionates the modulatory effects of D1 and D2 receptors on the neural machinery of a cognitive process.     

Distinct monoamine oxidase A and B populations in primate brain

Monoclonal antibodies specific for monoamine oxidase (MAO) A and MAO B, respectively, were used to localize these enzymes in primate brain. The reagents recognized different populations of neurons: those that recognized MAO A were located in cell groups containing catecholamines, including the substantia nigra, nucleus locus coeruleus, nucleus subcoeruleus, and the periventricular region of the hypothalamus, whereas those that recognized MAO B were observed in serotonin regions, including the nucleus raphe dorsalis and nucleus centralis superior. These data illustrate the physiological independence of MAO A and B and show that neurons may be specialized for their degradative as well as their synthetic functions.     

‘宁紫薯1号’花青素组分鉴定及其对大鼠高脂诱导肥胖的预防效果

【目的】紫甘薯是天然抗氧化物花青素的重要来源。鉴定‘宁紫薯1号’花青素(PSPC)组成成分,探索紫甘薯花青素对高脂诱导肥胖的预防效果并探讨其分子机制。【方法】通过液质联用技术分析紫甘薯新品种‘宁紫薯1号’的花青素组分;采用p H示差法测定制备的样品中总花青素含量。建立因高脂摄入过多导致机体产生营养性肥胖的动物模型,并灌胃不同剂量的紫甘薯花青素比较其预防肥胖效果。每周定时测量大鼠禁食6 h后的体重,通过试剂盒检测大鼠血清样品血糖(Glu)、甘油三酯(TG)、总胆固醇(TC)的含量,采用酶联免疫法检测瘦素(Leptin)含量。通过荧光定量PCR法(RT-PCR)检测下丘脑样品瘦素及其受体的m RNA表达,采用蛋白质免疫印迹法(Western blot)检测瘦素及其下游蛋白的信号表达。【结果】分离鉴定出11种PSPC组分,其中10种为已知的矢车菊素和芍药素酰基化衍生物,峰1为新发现的1种化合物。制备的花青素样品中矢车菊素-3-葡萄糖苷含量为17.4%。体重指标直接表明高剂量花青素组预防肥胖效果最好,为(358.2±20.1)g,中剂量组其次,为(371.6±16.3)g;效果最差的为低剂量组,体重为(384.0±7.2)g。紫甘薯花青素各剂量组大鼠血糖、甘油三酯和总胆固醇的3项血生化指标均趋于正常水平,有效预防肥胖的发生。血清瘦素检测结果显示,高脂组比对照组多释放65.50%;低剂量、中剂量和高剂量紫甘薯花青素组血清瘦素含量分别为(3.13±0.05)、(2.84±0.12)和(2.64±0.06)ng·m L~(-1),与高脂组相比均显著下降。同时不同剂量紫甘薯花青素组下丘脑中瘦素及瘦素受体的m RNA表达均显著高于高脂组。而高剂量紫甘薯花青素组可以调节下丘脑中瘦素信号,从而降低下游腺苷酸活化蛋白激酶α(AMPKα)的磷酸化。【结论】高剂量紫甘薯花青素可以通过调节下丘脑中leptin/AMPKα信号通路有效抑制高脂诱导肥胖的形成。     

Thyrotropin-releasing hormone: biosynthesis by rat hypothalamic fragments in vitro

Biosynthesis of thyrotropin-releasing hormone (L-pyroglutamyl-L-histidyl-L-proline amide) in vitro was studied. Rat hypothalamic fragments were incubated in Krebs-Ringer bicarbonate buffer that contained either (14)C-labeled proline, histidine, or glutamic acid (the three probable precursor amino acids,) and for control purposes each of 16 other naturally occurring amino acids. A number of labeled peptides were synthesized. With the use of synthetic thyrotropin-releasing hormone, detected by the Pauly reagent or with (125)1-labeled thyrotropin-releasing hormone as a marker, thin-layer chromatograms, paper electrophoresis, and carboxymethyl cellulose ion exchange chromatography revealed that only proline, histidine, and glutamic acid were consistently incorporated into peptides associated with the thyrotropin-releasing hormone region. This synthesizing activity was found in stalk median eminence, ventral hypothalamus. and dorsal hypothalamus but not in neural lobe or cerebral cortex. Because the biosynthetic peptide has identical properties with L-pyroglutamyl-L-histidyl-L-proline amide, it is probable that rat thyrotropin-releasing hormone is similar or identical to both bovine and porcine thyrotropin-releasing hormone and that the native material is present in the pyroglutamyl form in tissues.     

Distribution of ChAT in the Diencephalon of the One Day Chicken

The distribution of choline-acetyltransferase(ChAT)-like immunoreactive structure in the one day chicken diencephalons was examined by the method of streptavidin-Perosidase(SP)immunocytochemistry.It was found that ChAT immunoreactive neurons and fibers presented in many regions of one day chicken.Neural structure containing cholinergic neurons with varying density and immunoreactivity were the N.habexularis lateralis and N.habexularis medialis of the epithalamus,the N.rotundus,N.Ovoidalis,N.subrotundus and N.geniculatus lateralis of the thalamus,the hypothalamus,the N.decussationis supraopticae dorsalis of stratathalamus.There were also many varicose and nonvaricose fibers and terminals in the N.geniculatus lateralis and the hypothalamus.The results suggested that the diencephalons of one day chicken is richly innervated by the cholinergic neuron system.