家蚕色氨酸羟化酶 (TRH) 基因的克隆及表达特性分析

5-羟色胺 (5-hydroxytryptamine, 5-HT) 是生物界广泛分布的信号分子,涉及动物的重要行为。5-HT是色氨酸羟化酶 (Tryptophan hydroxylase, TRH) 将L-色氨酸羟化为5-羟-L-色氨酸,5-羟-L-色氨酸随即被多巴脱羧酶 (Aromatic L-amino acid decarboxylase, DDC) 脱羧而成。TRH作为5-HT合成的限速酶,在无脊椎动物神经调控中具有重要地位。鳞翅目昆虫中TRH的功能研究并不多。在家蚕中克隆了家蚕TRH (Bombyx mori TRH, BmTRH) 的cDNA序列1 667 bp,其中包含1 632 bp的开放读码框 (Open reading frame, ORF)。人类TPH或者果蝇TRH (Drosophila TRH, DmTRH) 与BmTRH有高度相似性,尤其BmTRH和DmTRH之间大多数氨基酸保守说明它们在系统发育上的密切关系并可能有相似功能。基因表达分析显示BmTRH主要表达于头部和中枢神经组织,免疫组织化学和Western blotting结果显示BmTRH只存在于神经组织中,即BmTRH可能仅参与家蚕的神经活动。此外,家蚕DDC (B. mori decarboxylase, BmDDC) 和蛋白具有TRH活性的苯丙氨酸羟化酶基因 (Phenylalanine hydroxylase, BmPAH) 也在中枢神经系统中有表达,暗示家蚕神经系统5-HT的合成与果蝇中不同,可能有两种不同的调控机制。     

五羟色胺和多巴胺与攻击行为的关联研究进展

五羟色胺（5-HT）和多巴胺（DA）是影响攻击行为的重要神经递质。参与这两种神经递质合成和分解、运输及信号转导等过程的物质均可能影响攻击行为,如影响5-HT作用的色氨酸、色氨酸羟化酶、单胺氧化酶、5-羟吲哚乙酸及5-HT转运体和5-HT受体;影响DA作用的多巴胺β羟化酶和儿茶酚胺邻位甲基转移酶以及DA转运体。未来攻击行为研究,应考虑色氨酸自身代谢、受体亚型及其他单胺类和儿茶酚胺类神经递质的影响。将肠道微生物纳入攻击行为研究也是未来研究的新方向。     

针刺对血和脑色氨酸浓度的影响

已知色氨酸是脑内5-羟色胺(5-HT)生物合成的前体,由血液转运入脑,但它在脑内的浓度低于色氨酸羟化酶(Tryptophan Hydroxylase)亲和力所需要的浓度。本文作者已证实,5-HT 能系统的正常功能是实现针刺镇痛的必需条件,针刺过程能激发脑内5-HT 能神经元的活动,使5-HT 生物合成增加,它的代谢产物5-羟吲(?)醋酸(5-HIAA)明显升高。那么,针刺镇痛过程是否也激发血脑色氨酸的转运功能,以保持脑内5-HT 能神经元正常活动的需要。本文针对这问题,在针刺镇痛后,分别测定血和脑色     

模拟微重力效应对果蝇运动及睡眠的影响

本研究旨在探讨利用模拟微重力效应研究微重力对果蝇运动及睡眠影响的可行性.通过研制能够在模拟微重力环境下实时监测果蝇行为的随机定位仪,监测短时间(3 d)模拟微重力处理过程中,及长时间(10 d、20 d、30 d)处理后雄蝇运动和睡眠的变化;选取受影响较显著的短时间处理组,研究模拟微重力效应对生物钟核心基因(period (per)、timeless(tim)、clock (clk)、cycle (cyc)、cryptochrome (cry))、神经递质多巴胺(dopamine,DA)和5-羟色胺(5-hydroxytryptamine,5-HT)关键合成酶(多巴脱羧酶、酪氨酸羟化酶、色氨酸羟化酶)的编码基因ddc、pale和trh表达水平及DA和5-HT含量的影响.结果显示:短时间暴露下,雄蝇夜晚的运动量增加、单位时间运动次数增加、睡眠时间和次数减少、生物钟基因tim、clk、cyc、cry及神经递质合成相关编码基因ddc、pale和trh的表达水平均显著上升;长时间处理后对雄蝇运动和睡眠的影响较小.本研究认为利用模拟微重力效应研究微重力对果蝇运动及睡眠的影响是可行的,相关研究结果对航天医学研究具有借鉴意义.     

昆虫5-羟色胺及其受体的研究进展

5-羟色胺（5-hydroxytryptamine, 5-HT）是昆虫体内一种重要的生物胺。5-HT在昆虫神经组织和非神经组织中均可合成,它可被5-HT转运体重吸收进入突触前结构中。5-HT通过结合特异性的G蛋白偶联受体在昆虫体内发挥不同的神经调控作用,调节昆虫主要的行为活动,比如取食、生物钟、聚集、学习和记忆等。昆虫体内5-HT受体有5种,分别为5-HT1A,5-HT1B, 5-HT2A,5-HT2B 和5-HT7。其中5-HT1A和5-HT1B偶联胞内cAMP的降低, 5-HT2A和5-HT2B偶联胞内Ca²⁺的释放, 5 HT7偶联胞内cAMP的升高。近年来,昆虫体内5-HT及其受体的研究有了很大的进展,昆虫体内越来越多的5-HT受体被克隆,并进行了功能和药理学性质分析。不同昆虫5 HT受体药理学性质存在差异,将为以5-HT受体为靶标,设计新型特异性杀虫剂提供理论基础。     

小鼠围生期应用抗生素可致雄性子鼠血清5-羟色胺水平升高及行为和肠道运动功能改变

本研究旨在探讨围生期母鼠使用抗生素对子代小鼠认知行为、肠道动力和外周血5-羟色胺(5-hydroxytryptamine,5-HT)水平的影响。在孕鼠分娩前一周至分娩后一周持续给予头孢克肟或青霉素处理,用三腔社交偏好实验、自我捋毛试验和高架十字迷宫实验检测4～10周龄子鼠行为学改变,检测子鼠的消化道动力,用ELISA检测血清5-HT水平,用RT-qPCR和Western blot检测子鼠结肠上皮组织钾/钠超极化激活环核苷酸门控通道2 (potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 2, HCN2)和色氨酸羟化酶1 (tryptophan hydroxylase 1, TPH1)表达水平。结果显示,与对照组相比,头孢克肟组雌性子鼠社交积极性显著提高,雄性子鼠无显著变化;雄性子鼠的结直肠动力和全肠道动力显著降低,盲肠净质量和相对体重占比显著增加,雌性子鼠无显著变化。与对照组相比,头孢克肟组和青霉素组雄性子鼠血清5-HT含量显著升高,雌性子鼠无显著变化。相比对照组,头孢克肟组子鼠结肠上皮HCN2表达水平显著下调,TPH1表达水平无显著变化。以上结果提示,围生期抗生素暴露可能影响子代神经发育和肠道功能,而且这种影响可能与外周血5-HT的变化相关且具有性别差异性。     

大鼠脑内5-羟色胺在应激性溃疡形成中的作用

通过神经化学和神经药理学的方法,在大鼠观察了冷冻加束缚应激性溃疡的形成过程中,脑内5-羟色胺(5-HT)的作用。结果如下:1.在应激过程中,脑内5-HT 及其主要代谢产物5-羟吲哚乙酸(5-HIAA)的含量明显升高,特别是5-HIAA 的含量随着应激时间的延长持续上升,说明5-HT 的代谢加快。2.脑内5-HT 或5-HIAA 含量在应激45min 时与溃疡指数呈明显的负相关,而在应激180min 时则与溃疡指数呈明显的正相关。3.侧脑室注射5-HT或其前体5-羟色氨酸(5-HTP),对应激性溃疡的形成呈双重作用,小剂量时减轻而大剂量时加重溃疡的形成。4.腹腔注射5-HT 合成阻断剂对氯苯丙氨酸(pCPA)可降低大鼠脑内5-HT 和5-HIAA 含量,使应激60min 鼠的溃疡形成加重,而使应激180min 鼠的溃疡形成减轻。以上结果提示,在大鼠的冷冻加束缚应激性溃疡的形成过程中,脑内5-HT 起着一定的作用,它很可能在应激早期减轻而在应激晚期加重溃疡的形成。     

家蚕非编码RNA在神经系统中的表达分析

【目的】非编码RNA(non-coding RNA,ncRNA)在家蚕Bombyx mori发育过程中具有重要调控作用。本研究旨在探索ncRNA参与家蚕神经系统发育的分子机理。【方法】采用实时荧光定量PCR技术对22个中等长度的ncRNA及3个ncRNA的邻近编码基因在家蚕幼虫神经系统中的表达水平进行检测。【结果】8个ncRNA(包括1个C/D box snoRNA,4个H/ACA box snoRNA和3个不能归类的ncRNA)在家蚕5龄幼虫神经组织和非神经组织中均有表达,且在胸腹神经中的表达量明显高于头部神经中的表达量。其中,snoRNA Bm-51,Bm-18和Bm-86在胸腹神经中的表达量分别是头部神经中的23,5和4.7倍。进一步研究发现,这3个内含子起源的ncRNA与其宿主基因在家蚕神经系统中的表达趋势一致,宿主基因在胸腹神经中的表达量也明显高于头部神经中的表达量。【结论】本研究筛选到的在家蚕不同神经部位存在差异表达的ncRNA,特别是在胸腹神经中高表达的ncRNA可能协同其邻近编码基因,参与家蚕神经发育或神经活动过程。该结果为研究ncRNA参与家蚕神经系统发育提供了分子依据,为从非编码RNA角度探索鳞翅目害虫防治提供了新的思路。     

酪氨酸羟化酶在成年大鼠胰腺中的定位

目的:酪氨酸羟化酶(tyrosine hydroxylase,TH)是儿茶酚胺类递质合成的限速梅,儿茶酚胺类递质对胰腺内分泌细胞的功能具有重要的调控作用,本研究拟探讨酪氨酸羟化酶(tyrosine hydroxylase,TH)在成年大鼠整个胰腺的具体定位和表达.方法:取雄性成年大鼠胰腺,冰冻组织切片,应用免疫荧光技术观察酪氨酸羟化酶在整个胰腺中的表达分布情况,并进一步运用免疫荧光双标技术鉴定酪氨酸羟化酶是否与胰岛素、胰高血糖素、生长抑素以及胰多肽分别共定位于β细胞;α细胞;δ细胞及PP细胞,进一步确定合成酪氨酸羟化酶确切的细胞类型.结果:①在胰腺腺泡细胞胞浆中存在酪氨酸羟化酶的阳性表达颗粒.②分布于胰腺外分泌腺的神经纤维和胰岛的神经纤维中都有酪氨酸羟化酶的表达.③酪氨酸羟化酶与胰岛的四种内分泌细胞所合成的肽之间均没有共定位关系.结论:在胰腺,酪氨酸羟化酶只存在于胰腺外分泌腺的腺泡细胞胞浆内以及胰腺中的神经纤维中,而胰岛四种内分泌细胞中没有酪氨酸羟化酶,说明胰腺儿茶酚胺类神经递质一方面由胰腺外分泌部的腺泡细胞合成,另一方面来源于神经末梢的释放,而胰岛细胞不能合成儿茶酚胺类递质;该结果为进一步研究胰腺内、外分泌部之间的关系和儿茶酚胺对胰腺分泌功能的调节提供形态学证据.     

色氨酸对应激小鼠行为学和脑内单胺类递质的影响

本文以小鼠为实验材料建模,依次进行了Morris水迷宫、跳台、强迫游泳实验。结果显示,应激可导致小鼠学习记忆能力下降,容易出现绝望情绪,下丘脑5-HT含量下降,NE含量上升,灌胃高低剂量色氨酸(100,200mg·kg-1 bw)均可显著提高小鼠学习记忆能力,缓和绝望情绪。低剂量色氨酸在增加下丘脑5-羟色胺(5-HT)含量和降低去甲肾上腺素(NE)含量方面效果更显著。结果表明,适量色氨酸可以有效改善应激小鼠相关行为学评分,其机制可能与下丘脑5-HT和NE的含量有关。     

Maternal and zygotic control of serotonin biosynthesis are both necessary for Drosophila germband extension.

In the accompanying paper, we report that Drosophila gastrulae genetically depleted for the 5-HT(2Dro) serotonin receptor or for serotonin show abnormal germband extension. In wild-type gastrulae, peaks of both the 5-HT(2Dro) receptor and serotonin coincide precisely with the onset of germband extension. Here, we assessed the genetic requirement for this peak of serotonin. We report the characterisation of the serotonin content of individual Drosophila embryos, progeny from flies heterozygous for mutations in genes that are involved in the serotonin synthesis pathway and include the GTP-cyclohydrolase, tryptophan hydroxylase and DOPA decarboxylase loci. The peak of serotonin synthesis at the beginning of germband extension appears strictly dependent upon the maternal deposition of biopterins, products of GTP-cyclohydrolase and cofactors of tryptophan hydroxylase and upon the zygotic synthesis of both tryptophan hydroxylase and DOPA decarboxylase enzymes. Mutant embryos with an impairment in this peak of serotonin synthesis die with a cuticular organisation which is also observed in embryos deficient for the 5-HT(2Dro) receptor. This characteristic cuticular phenotype is thus the hallmark of desynchronisation of the morphogenetic movements during gastrulation. Together, these findings provide additional support for the notion that serotonin, acting through the 5-HT(2Dro) receptor, is necessary for proper gastrulation.     

Role of the 5-HT7 Receptor in the Central Nervous System: from Current Status to Future Perspectives

Pharmacological and genetic tools targeting the 5-hydroxytryptamine (5-HT)7 receptor in preclinical animal models have implicated this receptor in diverse (patho)physiological processes of the central nervous system (CNS). Some data obtained with 5-HT7 receptor knockout mice, selective antagonists, and, to a lesser extent, agonists, however, are quite contradictory. In this review, we not only discuss in detail the role of the 5-HT7 receptor in the CNS but also propose some hypothetical models, which could explain the observed inconsistencies. These models are based on two novel concepts within the field of G protein-coupled receptors (GPCR), namely biphasic signaling and G protein-independent signaling, which both have been shown to be mediated by GPCR dimerization. This led us to suggest that the 5-HT7 receptor could reside in different dimeric contexts and initiate different signaling pathways, depending on the neuronal circuitry and/or brain region. In conclusion, we highlight GPCR dimerization and G protein-independent signaling as two promising future directions in 5-HT7 receptor research, which ultimately might lead to the development of more efficient dimer- and/or pathway-specific therapeutics.     

Cell-Autonomous Gβ Signaling Defines Neuron-Specific Steady State Serotonin Synthesis in Caenorhabditis elegans

Heterotrimeric G proteins regulate a vast array of cellular functions via specific intracellular effectors. Accumulating pharmacological and biochemical studies implicate Gβ subunits as signaling molecules interacting directly with a wide range of effectors to modulate downstream cellular responses, in addition to their role in regulating Gα subunit activities. However, the native biological roles of Gβ-mediated signaling pathways in vivo have been characterized only in a few cases. Here, we identified a Gβ GPB-1 signaling pathway operating in specific serotonergic neurons to the define steady state serotonin (5-HT) synthesis, through a genetic screen for 5-HT synthesis mutants in Caenorhabditis elegans. We found that signaling through cell autonomous GPB-1 to the OCR-2 TRPV channel defines the baseline expression of 5-HT synthesis enzyme tryptophan hydroxylase tph-1 in ADF chemosensory neurons. This Gβ signaling pathway is not essential for establishing the serotonergic cell fates and is mechanistically separated from stress-induced tph-1 upregulation. We identified that ADF-produced 5-HT controls specific innate rhythmic behaviors. These results revealed a Gβ-mediated signaling operating in differentiated cells to specify intrinsic functional properties, and indicate that baseline TPH expression is not a default generic serotonergic fate, but is programmed in a cell-specific manner in the mature nervous system. Cell-specific regulation of TPH expression could be a general principle for tailored steady state 5-HT synthesis in functionally distinct neurons and their regulation of innate behavior.     

Neuron-specific modulation by serotonin of regenerative outgrowth and intracellular calcium within the CNS of Helisoma trivolvis

We have investigated the cell-specific effect of serotonin (5-HT) on regenerating neurons within the adult central nervous system of the pond snail, Helisoma trivolvis. In culture, 5-HT arrests outgrowth of buccal neurons B19 but not neurons B5 (Haydon, McCobb, and Kater, 1984). After axotomy, neurons within the Helisoma nervous system typically exhibit profuse regenerative outgrowth. This study, on neurons within the CNS, shows that 5-HT selectively inhibits the outgrowth of specific identified neurons, and also causes significant elevations in intracellular calcium concentrations as measured by the calcium indicator dye, Fura-2. The outgrowth of neurons B19 and C1 was selectively inhibited when ganglia were incubated in 5 X 10(-5) M 5-HT. The outgrowth of buccal neurons B5, however, was not affected. Moreover, 5-HT caused significant transient elevations of calcium concentrations in neurons B19 over 30 minutes, but neurons B5 did not show any increases in calcium concentrations with the addition of 5-HT. These results suggest that the effect of 5-HT upon outgrowth of regenerating neurons may be due to an increase in the intracellular calcium concentration.     

Dopamine and sensory tissue development in Drosophila melanogaster

Dopamine is an important signaling molecule in the nervous system; it also plays a vital role in the development of diverse non-neuronal tissues in the fruit fly Drosophila melanogaster. The current study demonstrates that males depleted of dopamine as third instar larvae (via inhibition of the biosynthetic enzyme tyrosine hydroxylase) demonstrated abnormalities in courtship behavior as adults. These defects were suggestive of abnormalities in sensory perception and/or processing. Electroretinograms (ERGs) of eyes from adults depleted of dopamine for 1 day as third instar larvae revealed diminished or absent on- and off-transients. These sensory defects were rescued by the addition of L-DOPA in conjunction with tyrosine hydroxylase inhibition during the larval stage. Depletion of dopamine in the first or second larval instar was lethal, but this was not due to a general inhibition of proliferative cells. To establish that dopamine was synthesized in tissues destined to become part of the adult sensory apparatus, transgenic lines were generated containing 1 or 4 kb of 5' upstream sequences from the Drosophila tyrosine hydroxylase gene (DTH) fused to the E. coli beta-galactosidase reporter. The DTH promoters directed expression of the reporter gene in discrete and consistent patterns within the imaginal discs, in addition to the expected expression in gonadal, brain, and cuticular tissues. The beta-galactosidase expression colocalized with tyrosine hydroxylase protein. These results are consistent with a developmental requirement for dopamine in the normal physiology of adult sensory tissues.     

A tryptophan hydroxlyase 1 reporter that directs Cre recombinase extinguishable placental alkaline phosphatase expression in serotonergic (5-HT) neurons and peripheral tissues

The serotonergic (5-HT) system modulates many behaviors and has been implicated in psychiatric disorders, but the density of 5-HT processes has complicated analyses. We have used regulatory regions from the Tryptophan hydroxylase 1 (Tph1) gene to drive expression of LoxP-flanked placental alkaline phosphatase (PLAP) to generate the Tph1-Lox-PLAP reporter mouse line. In these mice, PLAP is expressed in the hindbrain raphe nuclei and in peripheral tissues known to express Tph1. Tph1 is expressed at low levels in neurons. While, in Tph1-Lox-PLAP mice, most PLAP-expressing neurons are monoaminergic, PLAP was expressed in only 5-10% of neurons expressing the predominant neuronal 5-HT biosynthetic enzyme Tph2, serotonin transporter (SERT) or aromatic amino acid decarboxylase (AADC). To test this reporter further, we examined the brains of mice carrying the anorexia (anx) mutation, in which increased overall density of 5-HT immunoreactivity had been previously observed at P21. PLAP-labeling of processes in anx/anx and anx/+ mice was reduced at P0. By P10, distribution of PLAP-labeled processes in anx/+ and +/+ cortices was indistinguishable, but differed markedly from that seen in the cortical layers of anx/anx mice. Thus, the Tph1-LoxP-PLAP reporter revealed a dosage sensitive role of the anx mutation in the early 5-HT system and later cortical layer-specific differences in 5-HT process distribution in anx/anx mice. Thus, the Tph1-LoxP-PLAP reporter provides a sensitive indicator for analyses of serotonergic cells in the brain and periphery.     

Substrate regulation of serotonin and dopamine synthesis in <Emphasis Type="Italic">Drosophila</Emphasis>

In Drosophila melanogaster, serotonin (5-hydroxytryptamine, 5-HT) is required for both very early non-neuronal developmental events, and in the CNS as a neurotransmitter to modulate behavior. 5-HT is synthesized, at least in part, by the actions of Drosophila tryptophan-phenylalanine hydroxylase (DTPH), a dual function enzyme that hydroxylates both phenylalanine and tryptophan. DTPH is expressed in numerous tissues as well as dopaminergic and serotonergic neurons, but it does not necessarily function as both enzymes in these tissues. Deficiencies in DTPH could affect the production of dopamine and serotonin, and thus dopaminergic and serotonergic signaling pathways. In this paper, we show that DTPH exhibits differential hydroxylase activity based solely on substrate. When DTPH uses phenylalanine as a substrate, regulatory control (end product inhibition, decreased PAH activity following phosphorylation, catecholamine inhibition) is observed that is not seen when the enzyme uses tryptophan as a substrate. These studies suggest that regulation of DTPH enzymatic activity occurs, at least in part, through the actions of its substrate.     

Role of tyrosine, DOPA and decarboxylase enzymes in the synthesis of monoamines in the brain of the locust

The metabolic transformation of tyrosine (TYR) by the decarboxylase and hydroxylase enzymes was investigated in the central nervous system of the locust, Locusta migratoria. It has been demonstrated that the key amino acids, 3,4-dihydroxyphenylalanine (DOPA), 5-hydroxytryptophan (5HTP) and tyrosine are decarboxylated in all part of central nervous system. DOPA and 5HTP decarboxylase activities show parallel changes in the different ganglia, but the rank order of the activity of TYR decarboxylase is different. Enzyme purification has revealed that the molecular weights of TYR decarboxylase and DOPA/5HTP decarboxylase are 370,000 and 112,000, respectively. The decarboxylation of DOPA by DOPA/5HTP decarboxylase is stimulated, whereas the decarboxylation of DOPA by TYR decarboxylase is inhibited in the presence of the cofactor pyridoxal-5'-phosphate. TYR hydroxylase could not be detected and 3H-TYR is found to be metabolised to tyramine (TA), but not to DOPA. The haemolymph contains a significant concentration of DOPA (120 pmol/100 microl haemolymph), and the ganglia incorporates DOPA from the haemolymph by a high affinity uptake process (K(M)=12 microM and V(max)=24 pmol per ganglion/10 min). Our results suggest that no tyrosine hydroxylase is present in the locust CNS and the DOPA uptake into the ganglia by a high affinity uptake process as well as the DOPA decarboxylase enzyme may be responsible for the regulation of the ganglionic dopamine (DA) level. Two types of decarboxylases exist, one of them decarboxylating DOPA and 5HTP (DOPA/5HTP decarboxylase), other decarboxylating TYR (TYR decarboxylase). The DOPA/5HTP decarboxylase enzyme present in the insect brain may correspond to the 5HTP/DOPA decarboxylase in vertebrate brain, whereas TYR decarboxylase is characteristic only for the insect brain.