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。     

5-HT_1受体各亚型mRNAs在大鼠脊髓不同节段背角和腹角的表达(英文)

为进一步了解5-HT受体在中枢感觉和运动机能中的作用,本研究利用反转录PCR方法比较了5-HT1A,5-HT1B,5-HT1D,5-HT1E和5-HT1F受体亚型mRNAs在脊髓不同节段的背角、腹角的表达。结果显示:5-HT1A,5-HT1B,5-HT1D受体亚型mRNA的表达水平在胸、腰、骶段脊髓的背角明显高于腹角;而颈段背角内的表达仅略高于腹角。脊髓内未检测到5-HT1E受体亚型;5-HT1F受体亚型在脊髓各节段的背角和腹角都有较高水平的表达。5-HT1受体家族各亚型mRNA在脊髓背角和腹角表达水平的差异可能提示它们在中枢感觉和运动功能中发挥不同的作用。     

5-HT_1受体亚型mRNAs在坐骨神经分支选择损伤模型大鼠脊髓背角的表达变化

为探讨脊髓内5-HT1受体亚型在神经病理性痛信息传递和调控中的作用,本研究利用反转录聚合酶链式反应(RT-PCR)技术观察了坐骨神经分支选择性损伤(SNI)模型大鼠脊髓背角内5-HT1A、5-HT1B、5-HT1D、5-HT1E和5-HT1F受体亚型mR-NAs表达变化。结果表明:损伤侧脊髓背角内5-HT1A受体亚型mRNA的表达水平在术后3d明显增高,7d达到高峰,随后开始下降,术后28d与正常对照相比仍有显著变化;5-HT1B受体亚型mRNA的表达水平也于术后3d开始显著增高,此后持续升高,至21d达到高峰,28d时有轻微下调,但高于对照水平;未观察到损伤侧脊髓背角内5-HT1D受体亚型mRNA表达水平的显著变化。5-HT1F受体亚型mRNA的表达水平于术后4d显著增高,此后一直维持该表达水平至28d。上述受体亚型在非损伤侧脊髓背角内的表达水平均无变化。在脊髓背角内未检测到5-HT1E受体亚型mRNA的表达。上述5-HT1受体亚型mRNA在SNI模型脊髓背角内具有不同的表达变化特点,提示不同的5-HT1各受体亚型在神经病理性痛信息的传递和调节中可能发挥着不同的作用。     

5-羟色胺系统参与学习记忆调节的研究进展

5-羟色胺（5-HT）系统在学习记忆中的调控作用越来越受到关注。5-HT能神经元是学习记忆研究最主要的神经元之一。5-HT释放作用于突触前/后膜各种亚型受体发挥其功能，尤其是对5-HT能神经元投射环路深入研究，将促进对学习记忆的探究。本文对5-HT受体、5-HT能神经元活动水平、5-HT能神经元投射环路参与学习记忆的相关研究进行阐述，为治疗学习记忆障碍提供理论依据。     

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。     

脊髓背角5-羟色胺纤维的来源和超微结构分布——HRP与免疫细胞化学结合法研究和免疫电镜观察

本文用HRP与免疫细胞化学结合法研究和免疫电镜观察进一步证实了脊髓背角内5-羟色胺(5-HT)能末梢的来源及其超微结构特征。结果表明:脊髓背角内5-HT能轴突末梢主要来自延髓中缝大核及腹侧网状结构。脊髓背角内5-HT免疫反应阳性结构见于Ⅰ层及Ⅱ层外带,为细的无髓纤维和有髓纤维。标记末梢与未标记末梢之间主要形成轴-轴突触(标记末梢可为突触前成分,也可是突触后成分),偶尔可见轴-树突触,未见非突触释放的形态学图象。根据其超微结构特征,我们推测脊髓背角内5-HT能系统不是以弥散的非突触方式影响有关结构,而是以突触方式直接或间接影响背角中间神经元的兴奋性,和直接抑制一级传入冲动而发挥其镇痛作用。     

5-羟色胺2A受体与疼痛

5-羟色胺(5-HT)是一种内源性的活性物质,5-HT具有多种受体亚型。在中枢可以通过2A受体引起抑制性神经递质γ-丁氨酸（GABA ）等的释放，从而发挥镇痛作用；在外周参与伤害性感受器的活化,促进伤害性信息的传递。组织损伤以及炎症状态下,血小板和肥大细胞即释放5-HT。5-HT2A受体可介导5-HT引起的疼痛,该受体可以作为开发外周镇痛药物的重要靶点。     

5-HT_(1A)受体亚型mRNA在坐骨神经分支选择性损伤模型大鼠脊髓和背根节中的表达变化(英文)

坐骨神经分支选择性损伤 (SNI)模型是一种新型神经病理痛模型。本实验用 SD雄性大鼠 ,分支结扎并切断左侧坐骨神经干的胫神经和腓总神经 ,保留腓肠神经分支 ,右侧仅暴露坐骨神经。术后 1、2、3、4、7、14、2 1和 2 8d,用 RT-PCR的方法对 5 -HT1 A受体 m RNA在腰髓的背角和背根神经节 (DRG)的表达水平进行检测。结果显示 ,5 -HT1 A受体 m RNA在损伤侧腰髓背角内的表达水平于 1d后开始升高 ,7d时达高峰 ,随后逐渐下降 ,但仍高于正常水平。其表达水平在对侧脊髓背角内没有明显变化。在损伤侧 DRG内 ,5 -HT1 A受体 m RNA的表达水平于 1d后开始增高 ,4d时达高峰 ,随后开始下降 ,但仍维持较高的表达水平 ;而损伤对侧 DRG内的 5 -HT1 A受体 m RNA的表达没有变化。上述结果提示 5 -HT1 A受体亚型可能在脊髓及外周伤害性信息的传递和调节中发挥着重要作用 ,本研究的结果为进一步了解 5 -HT1 A受体在神经病理性痛中的作用机制提供了依据。     

5-HT_(1A)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。     

刺激大鼠海马后PAG、NRM和脊髓背角5-HT的变化

目的:探讨刺激大鼠海马后导水管周围灰质(Periaqueductal central gray matter,PAG)、中缝大核(Nucleusraphes magnus,NRM)和脊髓背角5-HT的变化,分析海马在镇痛中的作用机制。方法:健康成年Wistar大鼠40只,随机分为4组。A组:正常对照组;B组:疼痛组;C组:谷氨酸钠组;D组:生理盐水组。四组动物均于2小时后处死,常规灌注冰冻连续切片,采用SABC免疫组化和计算机图像分析技术,检测PAG、NRM5-HT神经元阳性细胞个数及光密度及脊髓背角5-HT阳性纤维及终未的积分光密度等均值。结果:疼痛刺激后,PAG、NRM、脊髓背角内5-HT水平较正常显著增高(P<0.05),谷氨酸钠组则较疼痛组进一步增加。结论:海马兴奋后,激活内源性镇痛系统,使5-HT大量释放,参与镇痛。     

Metabotropic glutamate receptor 5 regulates excitability and Kv4.2-containing K⁺ channels primarily in excitatory neurons of the spinal dorsal horn

Metabotropic glutamate (mGlu) receptors play important roles in the modulation of nociception. Previous studies demonstrated that mGlu5 modulates nociceptive plasticity via activation of ERK signaling. We have reported recently that the Kv4.2 K(+) channel subunit underlies A-type currents in spinal cord dorsal horn neurons and that this channel is modulated by mGlu5-ERK signaling. In the present study, we tested the hypothesis that modulation of Kv4.2 by mGlu5 occurs in excitatory spinal dorsal horn neurons. With the use of a transgenic mouse strain expressing enhanced green fluorescent protein (GFP) under control of the promoter for the γ-amino butyric acid (GABA)-synthesizing enzyme, glutamic acid decarboxylase 67 (GAD67), we found that these GABAergic neurons express less Kv4.2-mediated A-type current than non-GAD67-GFP neurons. Furthermore, the mGlu1/5 agonist, (R,S)-3,5-dihydroxyphenylglycine, had no modulatory effects on A-type currents or neuronal excitability in this subgroup of GABAergic neurons but robustly modulated A-type currents and neuronal excitability in non-GFP-expressing neurons. Immunofluorescence studies revealed that Kv4.2 was highly colocalized with markers of excitatory neurons, such as vesicular glutamate transporter 1/2, PKCγ, and neurokinin 1, in cultured dorsal horn neurons. These results indicate that mGlu5-Kv4.2 signaling is associated with excitatory dorsal horn neurons and suggest that the pronociceptive effects of mGlu5 activation in the spinal cord likely involve enhanced excitability of excitatory neurons.     

Electrophysiological and pharmacological properties of GABAergic cells in the dorsal raphe nucleus

The dorsal raphe nucleus (DRN) is the origin of the central serotonin [5-hydroxytryptamine (5-HT)] system and plays an important role in the regulation of many physiological functions such as sleep/arousal, food intake and mood. In order to understand the regulatory mechanisms of 5-HT system, characterization of the types of neurons is necessary. We performed electrophysiological recordings in acute slices of glutamate decarboxylase 67–green fluorescent protein knock-in mice. We utilized this mouse to identify visually GABAergic cells. Especially, we examined postsynaptic responses mediated by 5-HT receptors between GABAergic and serotonergic cells in the DRN. Various current responses were elicited by 5-HT and 5-HT_1A or 5-HT_2A/2C receptor agonists in GABAergic cells. These results suggested that multiple 5-HT receptor subtypes overlap on GABAergic cells, and their combination might control 5-HT cells. Understanding the postsynaptic 5-HT feedback mechanisms may help to elucidate the 5-HT neurotransmitter system and develop novel therapeutic approaches.     

利用GAD67-GFP基因敲入方法显示小鼠脊髓内表达GFP的GABA能神经元的分布(英文)

γ-氨基丁酸 (GABA)是脊髓背角、前角内主要的抑制性神经递质。为了更好地观察脊髓背角内 GABA能神经元的形态和功能 ,本研究使用了两种谷氨酸脱羧酶 67-绿色荧光蛋白 (GAD67-GFP)基因敲入小鼠 ,并观察了敲入小鼠脊髓内的 GFP表达状况。用免疫荧光组织化学双标记方法显示脊髓内所有的 GF P阳性神经元基本上都呈 GAD67和 GABA阳性 ;GFP阳性神经元在脊髓背角的 ～ 层最为密集 ,背角深层内侧部及中央管周围呈中等密度分布 ,而在脊髓背角其它部位及前角则呈散在分布。脊髓内 GFP阳性神经元的分布与 GABA能神经元的分布一致。本文作者等还进一步在 GAD67-GFP敲入小鼠中观察了 GFP和神经元标志物神经元核蛋白 (Neu N )的共存状况。脊髓背角内 GFP阳性神经元分别占 、 和 层的 Neu N阳性神经元的 3 1.5 %、3 3 .3 %和 44 .7% ,与以往的 GABA免疫组化研究结果基本一致。本研究表明 GAD67-GFP基因敲入小鼠脊髓内的 GFP在GAD67启动子的调节下正确地表达于 GABA能神经元 ,该基因敲入小鼠可用于脊髓 GABA能神经元的形态学特征和生理学特性及其发育规律等方面的研究     

Expression of serotonin 5-HT2C receptors in GABAergic cells of the anterior raphe nuclei

We have used double in situ hybridization to examine the cellular localization of 5-HT2C receptor mRNA in relation to serotonergic and GABAergic neurons in the anterior raphe nuclei of the rat. In the dorsal and median raphe nuclei 5-HT2C receptor mRNA was not detected in serotonergic cells identified as those expressing serotonin (5-HT) transporter mRNA. In contrast, 5-HT2C receptor mRNA was found in most GABAergic cells, recognized by the presence of glutamic acid decarboxylase mRNA. Such 5-HT2C receptor-positive GABAergic neurons were mainly located in the intermediolateral and lateral portions of the dorsal raphe and lateral part of the median raphe. The present data give anatomical support to a previous hypothesis that proposed a negative-feedback loop involving reciprocal connections between GABAergic interneurons bearing 5-HT2A/2C receptors and 5-HT neurons in the dorsal raphe and surrounding areas. According to this model, the excitation of GABAergic interneurons through these 5-HT2C (and also 5-HT2A) receptors would result in the suppression of 5-HT cell firing.     

Descending projections from the rostral ventromedial medulla (RVM) to trigeminal and spinal dorsal horns are morphologically and neurochemically distinct

Neurons in the rostral ventromedial medulla (RVM) are thought to modulate nociceptive transmission via projections to spinal and trigeminal dorsal horns. The cellular substrate for this descending modulation has been studied with regard to projections to spinal dorsal horn, but studies of the projections to trigeminal dorsal horn have been less complete. In this study, we combined anterograde tracing from RVM with immunocytochemical detection of the GABAergic synthetic enzyme, GAD67, to determine if the RVM sends inhibitory projections to trigeminal dorsal horn. We also examined the neuronal targets of this projection using immunocytochemical detection of NeuN. Finally, we used electron microscopy to verify cellular targets. We compared projections to both trigeminal and spinal dorsal horns. We found that RVM projections to both trigeminal and spinal dorsal horn were directed to postsynaptic profiles in the dorsal horn, including somata and dendrites, and not to primary afferent terminals. We found that RVM projections to spinal dorsal horn were more likely to contact neuronal somata and were more likely to contain GAD67 than projections from RVM to trigeminal dorsal horn. These findings suggest that RVM neurons send predominantly GABAergic projections to spinal dorsal horn and provide direct input to postsynaptic neurons such as interneurons or ascending projection neurons. The RVM projection to trigeminal dorsal horn is more heavily targeted to dendrites and is only modestly GABAergic in nature. These anatomical features may underlie differences between trigeminal and spinal dorsal horns with regard to the degree of inhibition or facilitation evoked by RVM stimulation.     

Preproenkephalin mRNA is Expressed in a Subpopulation of GABAergic Neurons in the Spinal Dorsal Horn of the GAD67‐GFP Knock‐In Mouse

GABA (γ‐aminobutyric acid)ergic neurons in the spinal dorsal horn have been reported to be divided into distinctive populations, with different cotransmitters and neuropeptides. In this study, we examined the colocalization of enkephalin (ENK) mRNA with GABA in the spinal dorsal horn using the glutamic acid decarboxylase (GAD)₆₇‐green fluorescence protein (GFP) knock‐in mouse. Our approach was to perform in situ hybridization histochemistry to detect mRNA for preproenkephalin (PPE, the precursor protein for ENK), combined with immunohistochemistry for GFP to reveal GABAergic neurons. Quantitative analysis indicated that more than 44.4% (2967/6681) of GFP‐immunoreactive neurons showed signals for PPE mRNA in the spinal dorsal horn. While 53.9% (2967/5501) of PPE mRNA‐expressing neurons were immunoreactive for GFP. The double‐labeled neurons were observed throughout the spinal dorsal horn, although they had a preferential localization in superficial layers. The present results provide a detailed morphological evidence that ENK and GABA colocalized in a subpopulation of neurons in the spinal dorsal horn, which are likely to represent local inhibitory dorsal horn interneurons involved in the modulation of pain transmission. Anat Rec, 291:1334–1341, 2008. © 2008 Wiley‐Liss, Inc.     

Serotonin receptors 5-HT<Subscript>1A</Subscript> and 5-HT<Subscript>3</Subscript> reduce hyperexcitability of dorsal horn neurons after chronic spinal cord hemisection injury in rat

Spinal cord injury (SCI) results in abnormal pain syndromes in humans. In a rodent model of SCI, T13 spinal hemisection results in allodynia and hyperalgesia due in part to interruption of descending pathways, including serotonergic (5-HT) systems, that leads to hyperexcitability of dorsal horn neurons. To characterize further the role of 5-HT and 5-HT receptor subtypes 5-HT_1A and 5-HT₃ in neuronal activation after hemisection, we have examined the responsiveness of dorsal horn neurons to a variety of innocuous and noxious peripheral stimuli. Male Sprague-Dawley rats, 150–175 g, were spinally hemisected (n=40) at T13 and allowed 4 weeks for development of mechanical allodynia and thermal hyperalgesia. Animals then underwent electrophysiologic recording and the results were compared with those from sham controls (n=15). Evoked responses of convergent dorsal horn neurons (n=224 total) at L3–L5 to innocuous and noxious peripheral stimuli were characterized after administration of vehicle, 5-HT (25, 50, 100, and 200 μg), 5-HT (100 μg) in conjunction with the selective 5-HT_1A antagonist WAY 100135 (100 μg), the 5-HT₃ antagonist MDL 72222 (100 μg), the selective 5-HT_1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 150 μg), or the 5-HT₃ agonist 2-Me-5HT (75 μg), with or without pretreatment with antagonists; all treatments were delivered topically onto the cord adjacent to the recording electrode. In hemisected animals, increased responsiveness of convergent cells to all peripheral stimuli was observed bilaterally when compared to controls. No changes in ongoing background activity were present. In control animals, only the highest dose of 5-HT (200 μg) was sufficient to reduce evoked activity, whereas in hemisected animals a concentration-dependant decrease in response was observed. In hemisected animals, both 5-HT_1A and 5-HT₃ receptor antagonism reduced the effectiveness of 5-HT, restoring elevated evoked activity by up to 70% at the doses tested. Administration of 5-HT_1A and 5-HT₃ receptor agonists also decreased hyperexcitability, effects prevented by pretreatment with corresponding antagonists. These results demonstrate the development of denervation supersensitivity to 5-HT following SCI, corroborate behavioral studies showing the effectiveness of 5-HT in reducing allodynia and hyperalgesia after SCI, and contribute to a mechanistic understanding of the role of 5-HT receptor subtypes in chronic central pain. Electronic Publication