5-HT受体亚型在原代培养大鼠脊髓背角神经元的表达

为探讨脊髓背角内 5 -HT发挥作用的受体类型及其胞内信号转导机制 ,本研究首先利用免疫荧光技术对胎鼠脊髓背角神经元的产率进行了观察 ,再利用反转录 PCR方法观察了 5 -HT受体 14种亚型 m RNAs在原代培养神经元中的表达。原代培养的背角神经元生长状态良好 ,存活时间达到 14～ 2 1d。对培养的背角细胞用神经元特异性标志物—神经细胞核蛋白 (Neu N)进行免疫荧光检测的结果表明 ,神经元的产率超过 90 % ;用 PCR方法在培养的背角神经元中检测到了 5 -HT1 A、5 -HT1 B、5 -HT1 D、5 -HT1 F、5 -HT2 A、5 -HT2 C、5 -HT3、5 -HT4 、5 -HT5A、5 -HT5B、5 -HT6 和 5 -HT7等受体亚型 m RNAs的表达。但上述各受体亚型的表达水平存在差异 ,未检测到 5 -HT1 E和 5 -HT2 B受体亚型 m RNAs的表达。结果表明 ,本研究建立的实验方法可满意地获得原代培养脊髓背角神经元 ;这些神经元不但表达多种受体亚型 ,而且表达类型与以往在成年大鼠脊髓背角观察到的表达状况基本一致。上述结果为进一步开展 5 -HT作用机制的体外研究奠定了基础。     

慢性应激抑郁模型大鼠脑内5-HT1A和5-HT2A受体的变化

为了在5-羟色胺受体水平研究抑郁症的机制和三环类抗抑郁药物(TCAs)阿米替林的药理学机理,将24只SD雄性大鼠随机均分为三组,即对照组、抑郁组、阿米替林治疗组.应用[3H]8-OH-DPAT、[3H]Ketanserin作为标记配基,采用放射性配体受体结合法,分别测定大鼠海马5-HT1A受体、大脑皮层5-HT2A受体结合.结果显示抑郁大鼠海马 [3H]8-OH-DPAT 特异性结合(18.78±5.62 fmol/mg prot),较正常对照组(26.12±5.52fmol/mg prot )明显下降(P＜0.05).抑郁大鼠大脑皮层[3H]Ketanserin特异性结合(112.58±4.21fmol/mg prot),较正常对照组(86.28±4.24fmol/mg prot)明显增加( P＜0.05).阿米替林治疗3周后,可使抑郁大鼠海马5-HT1A受体与大脑皮层5-HT2A受体结合恢复正常.提示 海马5-HT1A受体结合下降、大脑皮层5-HT2A受体结合增加可能与抑郁症病因有关;海马5-HT1A受体、大脑皮层5-HT2A受体是阿米替林发挥抗抑郁作用的环节.     

5-HT1A受体亚型与P物质、Ⅰ型囊泡膜谷氨酸转运体和甘丙肽在大鼠背根神经节神经元中的共表达

为探讨5-HT1A受体亚型参与感觉信息调控的机制,本文利用免疫荧光组织化学双重染色技术观察了该受体亚型与P物质(SP)、I型囊泡膜谷氨酸转运体(VGLUT1)和甘丙肽(Gal)在大鼠背根神经节(DRG)神经元内的共存状况。结果表明:5-HT1A受体亚型阳性神经元占DRG神经元总数的46.2%,阳性神经元以大型及小型神经元为主。在DRG内观察到了5-HT1A/SP、5-HT1A/VGLUT1以及5-HT1A/Gal双标神经元。其中5-HT1A/SP双标神经元占5-HT1A受体亚型阳性神经元的34.6%,占SP阳性神经元的72.0%;5-HT1A/VGLUT1双标神经元占5-HT1A受体亚型阳性神经元的24.1%,占VGLUT1阳性神经元的18.5%;5-HT1A/Gal双标神经元占5-HT1A免疫阳性神经元的17.6%,占Gal免疫阳性神经元的63.8%。5-HT1A/SP和5-HT1A/Gal双标神经元主要为DRG的小型神经元,而5-HT1A/VGLUT1双标神经元主要为大、中型神经元。上述结果提示,5-HT1A受体亚型可能通过调节SP、谷氨酸以及Gal在初级传入终末及外周神经末稍的释放发挥其感觉信息的调节作用。     

5-HT_(1A)受体参与腹外侧眶皮层内5-HT诱发的抗伤害效应

为了明确5-HT参与腹外侧眶皮层(VLO)的抗伤害效应以及VLO内的5-HT1A受体与该效应之间的关系,本研究采用浅麻大鼠,以甩尾反射潜伏期(TFL)为指标,观察VLO内微量注射5-HT和选择性5-HT1A受体拮抗剂p-MPPI对TFL的影响。结果显示:VLO内微量注射5-HT(2.0、5.0、10.0μg)可剂量依赖性抑制辐射热诱发的TFL;而VLO内预先5min微量注射5-HT1A受体拮抗剂p-MPPI(0.2μg)可拮抗5-HT(10.0μg)诱发的抗伤害效应。本研究的结果提示5-HT参与VLO内的抗伤害效应,而该效应可能由5-HT1A受体介导。     

激活吻侧无颗粒岛叶皮层内5-HT_(1A)受体抑制福尔马林诱发的伤害性行为

目的:明确5-HT1A受体是否参与吻侧无颗粒岛叶皮层(rostral agranular insular cortex,RAIC)介导的抗炎性持续性痛效应。方法:采用行为药理学实验方法观察RAIC内微量注射选择性5-HT1A受体激动剂和拮抗剂对福尔马林诱发的伤害性行为(缩足反应)的影响。结果:RAIC内微量注射选择性5-HT1A受体的激动剂8-OH-DPAT(5.0μg/0.5μl)明显抑制福尔马林诱发的大鼠缩足反应;向RAIC内提前5min预先微量注射5-HT1A受体拮抗剂NAN-190(10μg/0.5μl)则可拮抗8-OH-DPAT(5.0μg/0.5μl)对缩足反应的抑制效应;RAIC内单独注射NAN-190对福尔马林诱发的大鼠缩足反应没有影响。结论:5-HT1A受体参与RAIC介导的抗炎性持续性痛效应。     

大鼠海马内5-羟色胺及其受体5-HT2A的表达

目的:观察5-羟色胺(5-HT)纤维和5-HT2A受体在大鼠海马CA1、CA2和CA3三个区域的分布特点.方法:用5-HT递质和5-HT2A受体特异性抗体的免疫组织化学显色以及图像处理与分析.结果:在海马内,抗5-HT2A受体的免疫反应阳性产物主要位于锥体细胞的细胞膜和树突,树突染色较深;5-HT2A受体的阳性胞体在C...     

PTSD样行为异常大鼠中缝背核5-HT1A受体的改变

目的观察创伤后应激障碍(PTSD)样行为异常大鼠中缝背核(DRN)神经元5-HT1A受体表达变化,为探讨PTSD的发病机制提供资料。方法成年健康雄性Wistar大鼠60只,随机分为连续单一刺激(SPS)模型1d、4d、7d组及正常对照组,采用免疫组化、Western blot方法检测PTSD样行为异常大鼠中缝背核神经元5-HT1A受体表达变化。结果 SPS刺激后1d大鼠中缝背核神经元5-HT1A受体表达水平开始逐渐升高,第4天高于第1天,第7天高于第4天。结论 PTSD样大鼠中缝背核神经元5-HT1A受体呈规律性过表达。     

Zacopride增强心肌内向整流钾电流(IK1)效应的受体和信号转导通路

目的:探讨5-HT4受体激动剂兼5-HT3受体阻断剂zacopride增强心肌内向整流钾电流(IK1)效应的受体和细胞信号转导机制.方法:应用全细胞膜片钳技术记录大鼠心室肌细胞膜IK1电流,分别观察10 μmol/L 5-HT4受体阻断剂 RS23597-190、10 μmol/L 5-HT3受体激动剂间氯苯双胍(m-CPBG)、5 μmol/L PKA 抑制剂 KT5720、5 μmol/L PKC 抑制剂GF109203x 和 5 μmol/L PKG抑制剂 KT5823对zacopride 增强IK1的影响.结果:10 μmol/L 5-HT4受体阻断剂RS23597-190本身可抑制IK1电流,在预先应用RS23597-190的基础上,1 μmol/L zacopride仍可明显激动IK1通道,使其内向电流(-100 mV)增强32.5% (P< 0.05).10 μmol/L 5-HT3受体激动剂m-CPBG对IK1电流无明显影响,也不能逆转1 μmol/L zacopride对IK1的增强效应(P> 0.05).此外,5 μmol/L PKA 阻断剂 KT5720能显著抑制1 μmol/L zacopride对IK1的增强效应(P< 0.05),而PKC 阻断剂GF109203x和PKG阻断剂KT5823则对1 μmol/L zacopride的效应无明显影响(P> 0.05).结论:Zacopride对 IK1的增强作用可能经由PKA介导的信号转导通路,而不依赖于5-HT3和5-HT4受体.     

大鼠前扣带回皮质5-HT_(1A,1B,2A,2C)受体亚型蛋白表达的细胞定位

目的:观察5-HT_(1A、1B)和5HT_(2A、2C)等受体在大鼠前扣带回皮质(anterior cingulated cortex,ACC)的分布及细胞定位。方法:将20只成年雄性Sprague-Dawley大鼠平均分成四组,每组5只分别进行四种亚型5-HT的免疫组织化学反应。动物经过灌注固定、脑冠状切片(25μm)后,以卵白素-生物素复合物法(ABC法)对切片进行染色、显微镜观察。结果:四种5-HT受体亚型在ACC都有分布。ACC内II-VI层均有5-HT_(1A)免疫阳性反应神经元的胞体和树突。5-HT_(1B)受体亚型定位在神经元胞体上,表达较弱。I-III层5-HT_(2A)免疫阳性反应较强,主要为标记的锥体神经元树突(Cg1区III层观察到部分阳性反应的神经元胞体);而V-VI层可见到明显的免疫阳性神经元胞体。5-HT2C受体定位于ACC之II-VI层神经元胞体上。结论:5-HT_(1A)、5-HT_(1B)、5-HT_(2A)和5-HT_(2C)等不同受体亚型在ACC内细胞上的定位有一定差异,提示它们介导5-HT对ACC神经元发生调节反应的细胞作用部位有选择性,因之它们介导的5-HT对ACC神经元的作用效果也可能不同。     

高频电刺激大鼠丘脑底核抑制背侧中缝核5-羟色胺表达

本研究探讨高频电刺激丘脑底核对大鼠背侧中缝核5-羟色胺(5-HT)表达的影响。实验动物分两组,刺激组给予高频电流(130Hz,100μA,60μs)刺激大鼠右侧丘脑底核,对照组大鼠右侧丘脑底核植入电极,但无电流输出。刺激结束后,用免疫组织化学方法染色背侧中缝核5-HT能神经元,检测背侧中缝核5-HT能神经元的数量和平均灰度值。结果显示电刺激组背侧中缝核5-HT阳性神经元数目与对照组比明显减少(t(13)=3.786,P=0.002),并且神经递质5-HT表达量减少,平均灰度值显著增高(t(13)=7.917,P<0.001)。本实验结果表明高频电刺激丘脑底核对背侧中缝核5-HT能神经元有抑制作用,在应用高频电刺激丘脑底核治疗Parkison病运动障碍时出现的情绪障碍可能与其有关。     

5-HT、生长抑素及 P物质在海洛因依赖大鼠直肠的定位和表达

目的 探讨海洛因依赖期间大鼠直肠内5-HT、生长抑素(SS)和P物质(SP)免疫反应(IR)细胞的形态学改变.方法 选取成年SD大鼠,分为海洛因依赖组(30只)、盐水对照组(30只)和正常对照组(6只),皮下注射海洛因建立大鼠海洛因依赖模型.取直肠组织用免疫组织化学SABC法及图像分析法进行研究.结果 与正常组及盐水对照组比较,海洛因依赖组大鼠直肠5-HT、SS及SP免疫反应细胞的细胞数均增多(P<0.05). 5-HT、SS、 SP-IR细胞免疫染色增强,图像分析显示,海洛因依赖期间大鼠直肠内5-HT、SS及SP 免疫反应细胞的平均灰度值均低于正常组及盐水对照组(P<0.05);其中5-HT、SS-IR细胞以31d时间组最低(P<0.05),SP-IR细胞以38d时间组最低(P<0.01).结论 海洛因依赖期间直肠5-HT、SS和SP-IR细胞的细胞数及平均灰度值均发生变化,提示5-HT、SS和SP合成和分泌增多.     

胃肠道5-HT内分泌细胞和肥大细胞与肝再生的关系

目的 探讨胃肠道及肝中5-HT免疫活性(5-HT-IR)内分泌细胞和肥大细胞(MC)与肝再生的关系.方法 150只SD大鼠随机分为对照组、手术对照(OC)组和实验组.实验组大鼠切除2/3肝(部分肝切除,PH),分别于术后不同时段取胃、小肠、肝组织.用免疫组织化学技术检测组织中5-HT-IR内分泌细胞和5-HT-IR MC,用甲苯胺蓝(TB)染色技术检测组织中MC.OC组除不切除肝叶外,其他同实验组.结果 1.实验组和OC组胃肠中5-HT-IR内分泌细胞数均于PH后2h较对照组下降(胃P<0.05,肠P<0.01),但实验组PH后6～72h极显著多于对照组(P<0.01),肝中未见5-HT-IR内分泌细胞.2.除PH后48～96h肝中MC数显著少于对照组(P<0.05)外,实验组和OC组胃肠及肝中MC数较对照组均无显著差异.3.在胃中5-HT-IR MC数于PH后3～6h和96h显著多于对照组(P<0.05),12～72h极显著多于对照组(P<0.01);在小肠中PH后2h和120h显著多于对照组(P<0.05).3～96h极显著多于对照组(P<0.01);肝中PH后48～72 h显著少于对照组(P<0.05).OC组胃肠和肝中5-HT-IR MC数较对照组均无显著差异;4.MC的5-HT阳性率在胃中于PH后2～96 h,在小肠中于PH后0.5～120 h逐渐增大,在肝中于1.5～24 h有所增加,48～72 h明显下降.OC组胃肠和肝中的阳性率较对照组均无明显变化.结论肝切除后的肝细胞增殖期内胃肠道5-HT-IR内分泌细胞和5-HT-IR MC数显著增多,两种细胞可能提供了在肝再生中起重要作用的5-HT.     

5-HT及其受体与神经免疫内分泌网络

神经免疫内分泌学是介于神经内分泌学和免疫学之间的一门边缘学科，学科内部各系统之间存在着双向联系，借助于系统之间共有的信息分子，如神经递质、激素和细胞因子及其受体的相互作用进行信息交流并相互影响而形成一个功能整体。5-羟色胺(5-hydroxytryptamine，5-HT）是一种重要的神经递质，近十几年来，人们发现它对免疫系统也有一定的作用。     

5-HT excites globus pallidus neurons by multiple receptor mechanisms

Anatomical and neurochemical studies indicated that the globus pallidus receives serotonergic innervation from raphe nuclei but the membrane effects of 5-HT on globus pallidus neurons are not entirely clear. We address this question by applying whole-cell patch-clamp recordings on globus pallidus neurons in immature rat brain slices. Under current-clamp recording, 5-HT depolarized globus pallidus neurons and increased their firing rate, an action blocked by both 5-HT(4) and 5-HT(7) receptor antagonists and attributable to an increase in cation conductance(s). Further experiments indicated that 5-HT enhanced the hyperpolarization-activated inward conductance which is blocked by 5-HT(7) receptor antagonist. To determine if 5-HT exerts any presynaptic effects on GABAergic and glutamatergic inputs, the actions of 5-HT on synaptic currents were studied. At 10 microM, 5-HT increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) but had no effect on both the frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs). However, 5-HT at a higher concentration (50 microM) decreased the frequency but not the amplitude of the mIPSCs, indicating an inhibition of GABA release from the presynaptic terminals. This effect was sensitive to 5-HT(1B) receptor antagonist. In addition to the presynaptic effects on GABAergic neurotransmission, 5-HT at 50 microM had no consistent effects on glutamatergic neurotransmission, significantly increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) in 4 of 11 neurons and decreased the frequency of mEPSCs in 3 of 11 neurons. In conclusion, we found that 5-HT could modulate the excitability of globus pallidus neurons by both pre- and post-synaptic mechanisms. In view of the extensive innervation by globus pallidus neurons on other basal ganglia nuclei, this action of 5-HT originated from the raphe may have a profound effect on the operation of the entire basal ganglia network.     

大鼠孤束核内5-HT和内脏神经初级传入终末与向臂旁核投射神经元的联系-激光共聚焦扫描显微镜研究

本文在激光共聚焦扫描显微镜下观察了内源性5-HT终末、Ⅸ、X脑神经初级传人终末在孤束核内与向臂旁核投射神经元之间的联系.分别向臂旁外侧核微量注射四甲基罗丹明,Ⅸ、X脑神经干内微量注射生物素化葡聚糖胺,以免疫荧光和荧光组织化学方法显示5-HT、内脏神经初级传人终末和孤束核内的四甲基罗丹明逆行标记神经元.结果发现,大量罗丹明逆行标记细胞(红色)主要位于注射侧孤束核的的内侧、小细胞亚核及连合核.同部位也同时观察到5-HT能阳性终末(蓝色)和内脏神经初级传人终末(绿色).两种终末的终扣和膨体紧靠逆行标记神经元的胞体和树突或与其重叠,这些可能是突触连接部位的接触点.本文结果提示中枢内的下行性5-HT纤维可能直接作用于孤束核内向臂旁核投射的神经元且通过与内脏神经传人终末在此处的汇聚,而产生新的神经效应.     

Time-course of alterations in morphine-induced analgesia and nociceptive threshold following medullary raphe lesions

Electrolytic lesions which included major portions of the nucleus raphe magnus produced alterations in nociceptive threshold and morphine-induced analgesia. These effects were highly dependent on the time at which measurements were made following the lesioning procedure. Seven days or less after placement of the lesions there was a pronounced decrease in nociceptive threshold, but no effect on the capacity of morphine to induce antinociception. However, the nociceptive threshold exhibited recovery by 21–28 days, during which time there was a gradual, but profound, decrease in the capacity of morphine to induce analgesia. At longer times after the placement of lesions (28–35 days), the nociceptive threshold, assessed using the tail-flick test, became elevated concomitant with a recovery of morphine's analgesic potency.The differential time course of the lesion-induced attenuation of morphine analgesia and decrease in nociceptive threshold suggests that these effects result from the destruction of separate neuronal systems. Thus, the lesion-induced decrease in nociceptive threshold probably results from destruction of raphespinal serotonergic neurons while the attenuation of morphine analgesia is most likely an indirect result of damage to non-serotoninergic raphe-spinal neurons. Finally, the failure of acute raphe lesions to alter morphine analgesia indicates that the neurons found in the raphe magnus are not directly involved in mediating the antinociceptive actions of opiates.     

5-HT在人正常淋巴结及淋巴瘤中表达的研究

目的：通过对5－羟色胺（Serotonin,5-HT)在人淋巴结和淋巴瘤中表达的检测，寻找神经内分泌系统与免疫系统之间功能双向调节的免疫形态学依据，并探讨5－HT在正常状态和病理状态下对免疫系统的影响和作用。以及其在淋巴瘤发生过程中可能的作用。方法：运用免疫组织化学SABC法，以5－HT为探针对人正常或反应性增生的淋巴结（以下称正常淋巴结）35例及淋巴瘤30例进行检测，所得结果用统计学卡方检验法进行比较分析。结果：人正常淋巴结33／35例阳性表达，淋巴瘤中见20／30例呈阳性表达,胡比正常淋巴结表达强度明显降低，两者差异有显著性（P＜0．05），阳性表达主要分布在副皮质区的T淋巴细胞，淋巴滤泡生发中心细胞以及巨噬细胞等部位，结论：5－HT是神经内分泌免疫网络中共同的生物语言，其对免疫系统及神经内分泌系统起着双向调节的作用，并且可能在淋巴瘤的发生中发挥着一定的作用。     

Comparison of the effects of 5-HT<Subscript>1A</Subscript> and 5-HT<Subscript>4</Subscript> receptor activation on field potentials and epileptiform activity in rat hippocampus

The effects of serotonin (5-HT) as well as 5-HT_1A and 5-HT₄ receptor agonists, (±)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (8-OH-DPAT) and zacopride, respectively, on population spikes evoked by electrical stimulation and on spontaneous epileptiform activity were investigated in CA1 area of hippocampal slices. Spontaneous epileptiform activity was recorded from slice in a nominally Mg²⁺-free medium. While 5-HT application resulted in a decrease of population spikes evoked in standard incubation conditions, in accordance with earlier studies, it exerted two opposite effects on epileptiform activity. The early inhibitory effect was mimicked by 8-OH-DPAT while the later, excitatory, by zacopride. The application of 8-OH-DPAT decreased, and that of zacopride increased, the amplitude of population spikes. A comparison of the dose-dependence of the excitatory and inhibitory effects of serotonergic agonists on the amplitude of the population spike and on the frequency of epileptiform discharges indicated that the latter is a more sensitive measure of the activation of 5-HT_1A and 5-HT₄ receptors than the former. Thus, spontaneous epileptiform activity recorded in a nominally Mg²⁺-free slice medium represents a convenient model for investigation of hippocampal neuronal reactivity to the activation of various 5-HT receptor subtypes. Electronic Publication     

Expression of serotonin2A receptors in Purkinje cells of the developing rat cerebellum

Previous physiological and pharmacological studies have shown that the serotonin_2A (5-HT_2A) receptor is involved in cerebellar functions. However, the expression of 5-HT_2A receptors in the developing cerebellum has not been elucidated to date. In the present immunohistochemical study, we examined developmental changes of the distribution of 5-HT_2A receptors in Purkinje cells of the rat cerebellum from embryonic day 18 (E18) to postnatal day 21 (P21). The weak immunoreaction to 5-HT_2A receptors was found in the deep cerebellar nuclei on E19. In the cerebellar cortex of the hemisphere and the posterior vermis, somata of Purkinje cells became weakly immunoreactive on P0. With the dendritic elongation and arborization, the immunoreaction appeared in the proximal parts of Purkinje cell dendrites. Distal parts of the dendrites became immunoreactive after P12, and were strongly immunolabeled by P21. The present study may provide a structural basis to investigate the roles of 5-HT_2A receptors during the cerebellar development.