5-羟色胺3A受体在成年小鼠海马中间神经元中的分布

目的:利用成年5-HT3AR-BACEGFP转基因小鼠,研究5-羟色胺3A受体(5-HT3AR)在海马中间神经元中的分布情况。方法:成年5-HT3AR-BACEGFP转基因小鼠经心脏灌注固定后,利用免疫荧光双标记方法,并结合激光共焦显微镜扫描技术,观察5-HT3AR在成年5-HT3AR-BACEGFP转基因小鼠海马中不同中间神经元内的表达和分布情况。结果:5-HT3AR在成年小鼠整个海马中都有分布,且主要在CA1区、CA2/CA3区和齿状回有大量5-HT3AR免疫阳性细胞;激光共聚焦显微镜下观察到5-HT3AR阳性产物在细胞核、细胞浆和树突上均有表达;免疫荧光双标实验结果表明5-HT3AR阳性产物在CB(calbindin),CR(calretinin),Reelin,Som(somatostatin),NPY(neuropeptide Y)和VIP(vasoactive intestinal peptide)免疫阳性神经元中表达,但在PV(parvalbumin)免疫阳性神经元中不表达。定量结果显示:几乎所有的VIP阳性神经元均表达5-HT3AR阳性,约3/4的CR阳性神经元表达5-HT3AR,约1/2的CB、Reelin、NPY和Som阳性神经元表达5-HT3AR阳性;约1/4的5-HT3AR阳性神经元中表达Reelin,1/5的表达Som,5-HT3AR/CB和5-HT3AR/CR双标神经元各占5-HT3AR阳性神经元的1/10左右。结论:5-HT3AR-BACEGFP转基因小鼠能够作为研究海马中5-HT3AR功能及其在中间神经元中的作用机制研究的工具鼠。     

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-HT及其2A受体在大鼠丘脑前核的表达

目的研究5-羟色胺(5-HT)及其5-羟色胺2A受体(5-HT2AR)在大鼠丘脑前核的表达,探讨两者参与学习记忆的形态学依据。方法免疫组织化学ABC法观察5-HT及5-HT2AR在丘脑前核内的表达情况。包埋前免疫电镜技术观察丘脑前核群的5-HT能投射纤维终末。结果免疫组化结果显示:在大鼠丘脑前核群的前、中、后部均可见阳性的5-HT能神经元及大量串珠状的投射纤维终末,其中背侧核(AD)的神经元着色较深,胞体较大,纤维密集,平均光密度值(A值)与腹侧核(AV)的比较差异显著(P0.05);5-HT阳性反应产物主要定位于胞浆内,胞核不着色。包埋前免疫电镜显示:阳性5-HT能轴突终末与树突形成非对称性的轴-树突触。在AD、AV内可见黄色的5-HT2AR阳性神经元,其中AD的神经元胞体较大,着色较AV深,阳性产物灰度值二者比较差异显著(P0.05);阳性产物主要定位于神经元胞膜,胞核不着色。结论 5-HT和5-HT2AR在大鼠丘脑前核表达,在AD、AV的表达强度不同。     

5-HT_5与5-HT_6受体

5-HT_5受体又分为5-HT_(5A)及5-HT_(5B)两种亚型受体,5-HT_(5A)受体主要存在于小脑、大脑皮层、海马等脑区;5-HT_(5B)受体仅在海马CA;区、缰核、背侧缝际核发现,该受体与麦角胺(Ergo-tamine)、5-羧基酰氨基色胺(5-Carboxamidotryptamine,5-CT)、5-HT、美西麦角(Methysergide)等配体亲和力较大,可能与5-HT_(1D)受体有某种功能联系。5-HT_(6)受体主要在纹状体、边缘脑和大脑皮层等脑区表达,这类受体与三环类抗精神病药及抗抑郁药有高亲和力反应,提示它参与某些精神疾病的发生与治疗。     

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。     

多重脑震荡大鼠5-羟色胺能神经纤维变化

目的:研究多重脑震荡(MCC)与大鼠认知记忆相关脑区5-羟色胺(5-HT)能神经纤维变化的规律,以探讨MCC大鼠认知行为障碍的神经生物学基础。方法:应用自制单摆式机械打击装置构建MCC大鼠模型,免疫组织化学及半定量方法研究MCC后大鼠5-HT能神经纤维的变化。结果:前额叶皮质、中隔区、丘脑、海马CA1和CA3区5-HT能神经纤维免疫反应阳性物在伤后2d组反应达高峰,杏仁体、海马CA2和CA4区在伤后4d组免疫反应了阳性最强;顶叶皮质、梨状皮质及伏隔核内侧在伤后8d组反应最强。5-HT纤维密度在中隔区和伏隔核内侧以伤后2d最高,杏仁体区以伤后1d最高,大脑皮质和丘脑5-HT纤维密度在伤后各损伤组与对照组相比无显著性差异。结论:MCC可引起与认知功能有关脑区的5-HT表达增高,5-HT的高表达可能由脑损伤后神经元过度兴奋所引起的神经递质紊乱造成,而这种紊乱可能影响大鼠的认知功能。     

大鼠前庭神经核复合体内5-HT能终末与表达5-HT1A受体的前庭-臂旁核投射神经元之间的联系

目的 观察大鼠前庭神经核复合体(VNC)内5-羟色胺(5-HT)样阳性终末与表达5-HT1A受体(5-HT1A R)的前庭-臂旁核投射神经元之间的联系.方法 运用逆行束路追踪和免疫荧光组织化学染色相结合的双重标记技术,在激光共焦显微镜下观察.结果 将四甲基罗达明(TMR)注入臂旁核后,在双侧VNC的各个核团内均可观察到许多TMR逆标神经元,但以同侧为主.免疫荧光组织化学染色结果显示,在前庭内侧核(MVe)、前庭下核(SpVe)、前庭上核(SuVe)、前庭外侧核(LVe)、X核以及Y核的一些区域内,许多神经元表达5-HT1A R样免疫阳性,并可观察到大量5-HT样阳性纤维和终末.激光共焦显微镜下可进一步观察到一些TMR逆标神经元同时呈5-HT1A R样免疫阳性,且有部分5-HT样阳性终末与TMR/5-HT1A R双标神经元的胞体或树突形成密切接触.结论 提示5-HT可能通过5-HT1A R对前庭神经核复合体-臂旁核间的信息传递发挥调控作用.     

大鼠纹状体边缘区内5-HT_(2A)受体的分布──原位杂交和免疫组织化学研究

已知纹状体边缘区有密集的 5 -HT纤维及终末分布。为了观察 5 -HT2 A受体 m RNA是否在大鼠纹状体边缘区表达 ,从基因分子水平和细胞水平进一步证明大鼠纹状体边缘区能否合成 5 -HT2 A受体 ,用地高辛标记的寡核苷酸探针进行原位杂交 ,研究了大鼠纹状体边缘区的 5 -HT2 A受体 m RNA的表达及分布。原位杂交结果发现 5 -HT2 A受体 m RNA阳性杂交信号在纹状体的分布不均匀 ,尾壳核只有少量中等大小的阳性胞体 ,苍白球也只有少量较大的阳性胞体 ,而在尾壳核和苍白球之间的边缘区部位则可见到许多中等大小的梭形阳性神经元胞体 ,并呈现密集的带状分布。免疫组织化学结果观察到 5 -HT受体阳性神经元胞体在纹状体的分布与原位杂交结果一致。本实验结果推测 ,大鼠纹状体边缘区神经元可以合成 5 -HT2 A受体 ,具有接受和整合 5 -HT神经递质的功能     

5-HT受体亚型mRNAs在大鼠背根神经节和三叉神经节的表达—原位杂交组织化学法研究

应用原位杂交组织化学技术观察了大鼠背根节和三叉神经节内 5 -HT2 、5 -HT3 、5 -HT4、5 -HT5 、5 -HT6 和 5 -HT7受体亚型 m RNAs的表达。结果显示 :在背根节和三叉神经节中均观察到了 5 -HT2 A、5 -HT3 、5 -HT4和 5 -HT7受体亚型 m RNAs阳性细胞 ;两种神经节内上述受体亚型 m RNAs阳性细胞的百分比不同。 5 -HT3 受体亚型 m RNA阳性细胞的百分比最高 ,分别为 3 0 .4% (背根节 )和 3 2 .8% (三叉神经节 )。约 17.2 %和 14 .6 %的背根节和三叉神经节细胞分别呈 5 -HT2 A受体 m RNA阳性 ;而 5 -HT4和 5 -HT7受体亚型 m RNAs阳性细胞在此二节中的百分比分别为 14 .9%、2 4.7%和 11.2 %、9.8%。各受体亚型 m RNA阳性细胞均以中、小型节细胞为主。在各神经节内均未观察到 5 -HT2 B、5 -HT2 C、5 -HT5 和 5 -HT6 受体 m RNA的表达。上述结果提示 5 -HT2 A、5 -HT3 、5 -HT4和 5 -HT7受体亚型在外周伤害性感受中可能发挥着重要的作用     

成年大鼠Pre-B(o)tzinger复合体5-羟色胺样阳性神经纤维与神经激肽1受体样阳性神经元的关系

位于新生和成年哺乳动物延髓腹外侧区的Pre-B(o)tzinger复合体(pre-Botzinger complex,PBC)被认为是呼吸节律产生中枢,神经激肽1受体(neurokinin-1 receptor,NK1R)是PBC神经元的主要标记物.5-羟色胺(5-hydroxytryptamine,5-HT或serotonin)作为一种重要的神经递质,可通过其受体调节呼吸中枢的节律性变化.本研究采用免疫荧光组织化学和免疫电镜双重标记技术,在激光共聚焦显微镜和电子显微镜下观察了大鼠PBC内5-HT样免疫阳性神经纤维与NK1R样免疫阳性神经元或NK1R免疫阴性神经元之间的关系,旨在为5-HT参与PBC神经元呼吸节律的产生和调控提供形态学依据.共聚焦显微镜结果显示:正常大鼠PBC内可观察到一定数量的5-HT样阳性纤维和终末,且有少量的阳性纤维和终末与PBC内NK1R样阳性神经元的胞体或树突形成密切接触.在电镜下可进一步观察到:5-HT样阳性纤维终末可与NK1R样阳性神经元的树突形成直接接触,但未观察到典型的突触联系.5-HT样阳性纤维终末可与NK1R免疫阴性的树突形成非对称性突触.本实验结果提示:5-HT可能通过旁分泌的方式或通过中间神经元间接调节PBC内NK1R样阳性神经元的活动,以实现对中枢呼吸节律的调控.     

Localization of 5-HT2A, 5-HT3, 5-HT5A and 5-HT7 receptor-like immunoreactivity in the rat cerebellum

Although serotonin (5-hydroxytryptamine, 5-HT) is known to exert a modulatory action on cerebellar function, our current knowledge of the nature of receptor subtypes mediating serotonergic activity in this part of the brain remains fragmentary. In this study, we report the presence and distribution of 5-HT3, 5-HT5A and 5-HT7 receptor-like immunoreactivity in the rat cerebellum using immunofluorescence histochemistry. 5-HT3 immunoreactivity was found in fibers sparsely distributed throughout the cerebellum. Most of them were seen in the cerebellar cortex as fine varicose 5-HT3-positive axonal processes. 5-HT5A immunoreactivity, on the other hand, was observed in neuronal somata of the cerebellar cortex and deep cerebellar nuclei. Based upon cell morphology and the use of cell-specific markers, Purkinje cells, molecular layer interneurons and Golgi cells were found to be 5-HT5A immunopositive. In addition, the use of cell-specific markers allowed us to identify previously reported large 5-HT2A-positive cells in the granular layer as being Golgi cells. Finally, 5-HT7 immunoreactivity was observed only in Purkinje cells. Corroborating previous radioligand-binding, in situ hybridization and immunohistochemical studies, our data relate serotonin receptor subtypes to specific cerebellar cell types and may consequently contribute to the elucidation of serotonergic actions in the cerebellum.     

5-HT4, 5-HT6, 5-HT7; molecular pharmacology of adenylate cyclase stimulating receptors

Throughout the nervous system the receptors for the neurotransmitter serotonin form an unusually diverse set. Receptor-mediated responses to serotonin utilize a large collection of biochemical second messenger pathways, such as the stimulation or inhibition of adenylate cyclase activity, the hydrolysis of phosphoinositides, the mobilization of calcium and direct gating of ion channels. This diversity of structure and activity has been substantiated by the application of molecular cloning techniques which have now yielded at least 15 distinct molecular entities. The most recent subset of receptors for serotonin to be cloned are those that couple to the stimulation of adenylate cyclase activity. These subtypes: 5-HT₄, 5-HT₆ and 5-HT₇, although sharing a common signal transduction pathway, are remarkably divergent in their amino acid sequences, distribution in the brain and pharmacological properties. This digression from the expected relationships of receptor subtypes based on other serotonin receptors, as well as other biogenic amino receptor families, is unexpected and raises many questions about the extreme diversity of this signaling system.     

5-HT activates vagal afferent cell bodies in vivo: role of 5-HT2 and 5-HT3 receptors

Occipital artery (OA) injections of 5-HT elicit pronounced reductions in heart rate and mean arterial blood pressure (MAP) in urethane-anesthetized rats by activation of vagal afferent cell bodies in the ipsilateral nodose ganglion. In contrast, internal carotid artery (ICA) and i.v. injections elicit similar cardiovascular responses by activation of peripheral vagal afferent terminals. The aim of this study was to examine the roles of 5-HT3 and 5-HT2 receptors in the 5-HT-induced activation of vagal afferent cell bodies and peripheral afferent terminals in urethane-anesthetized rats. OA, ICA and i.v. injections of 5-HT elicited dose-dependent reductions in heart rate and MAP that were virtually abolished after i.v. administration of the 5-HT3 receptor antagonists, MDL 7222 or ICS 205-930. The responses elicited by the OA injections of 5-HT were markedly diminished after i.v. injection of the 5-HT2 receptor antagonists, xylamidine or ketanserin, whereas the responses elicited by i.v. or ICA injections of 5-HT were not affected. The present findings suggest that (1) 5-HT3 and 5-HT2 receptor antagonists gain ready access to nodose ganglion cells upon i.v. administration, and (2) functional 5-HT3 and 5-HT2 receptors exist on the cell bodies of vagal afferent neurons mediating the cardiovascular responses elicited by OA injections of 5-HT. These findings also support a wealth of evidence that 5-HT3 receptors exist on the peripheral terminals of vagal afferents, and although they do not discount the possibility that 5-HT2 receptors exist on peripheral vagal afferent terminals, it appears that activation of these receptors does not have pronounced effects on 5-HT3 receptor activity on terminals that mediate the hemodynamic responses to 5-HT.     

Identification of a 5-HT1 recognition site in human brain membranes different from 5-HT1A, 5-HT1B and 5-HT1C sites

In human caudate and cortex membranes, [3H]serotonin ([3H]5-HT) labels 5-HT1A and 5-HT1C recognition sites which show nanomolar affinity for 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)-tetralin) and mesulergine respectively, whereas no 5-HT1B binding could be identified. However, the majority of the sites labelled by [3H]5-HT (greater than or equal to 60% in cortex, 90% in caudate) are different from 5-HT1A, 5-HT1B and 5-HT1C sites. Competition experiments were performed in human caudate membranes incubated with [3H]5-HT in the presence of 100 nM 8-OH-DPAT and 100 nM mesulergine. Under those conditions, [3H]5-HT labelled an apparently homogeneous population of 5-HT1-like sites which display nanomolar affinity for tryptamines (5-carboxamido-tryptamine, (5-CT) greater than 5-HT greater than or equal to 5-methoxytryptamine (5-MeOT) greater than tryptamine) and some ergolines (metergoline greater than methysergide). In contrast, these sites showed low affinity for drugs with high affinity and/or selectivity for 5-HT1A (8-OH-DPAT, buspirone), 5-HT1B (21-009, RU 24969), 5-HT1C (mesulergine, mianserin) and 5-HT2 sites (ketanserin, cinanserin). The pharmacological profile of these sites is different from that of 5-HT1A, 5-HT1B, 5-HT1C, 5-HT2 and 5-HT3 sites but is consistent with the pharmacology of a 5-HT1-like receptor. It is very similar to that of the 5-HT1D site recently described in bovine brain by Heuring and Peroutka.     

5-HT reuptake and 5-HT2 receptors modulate capsaicin-evoked hypothermia in rats

Numerous studies support a role for the endogenous 5-hydroxytryptamine (5-HT) system in the hypothermic effect of capsaicin. None of those studies, however, selectively delineate a role for 5-HT reuptake or 5-HT receptors in this regard. In the present investigation, we determined if the blockade of 5-HT reuptake or the activation of 5-HT_1A or 5-HT₂ receptors modulates capsaicin-evoked hypothermia. The administration of capsaicin (0.2–1 mg/kg, i.m.) produced dose-related hypothermia. Fluoxetine (10 mg/kg, i.p.), a selective serotonin reuptake inhibitor (SSRI), did not affect body temperature. For combined administration, pretreatment with fluoxetine (10 mg/kg, i.p.) significantly attenuated the hypothermia caused by capsaicin (0.5 and 1 mg/kg, i.m.). For the 5-HT receptor experiments, we pretreated rats with either WAY 100635, a 5-HT_1A receptor antagonist, or mianserin, a 5-HT₂ receptor antagonist, and then administered a fixed, hypothermic dose of capsaicin (1 mg/kg, i.m.). WAY 100635 (1 mg/kg, s.c.) administration did not affect capsaicin-evoked hypothermia. This indicates that 5-HT_1A receptor activation does not play a major role in the hypothermic effect of capsaicin. In contrast, pretreatment with mianserin (10 mg/kg, i.p.) enhanced the hypothermic effect of capsaicin (1 mg/kg, i.m.). The present data reveal that capsaicin-evoked hypothermia in rats is attenuated by the blockade of 5-HT reuptake and enhanced by the antagonism of 5-HT₂ receptors.     

5-HT1A, but not 5-HT2 and 5-HT7, receptors in the nucleus raphe magnus modulate hypoxia-induced hyperpnoea

Aim: In the present study, we assessed the role of 5‐hydroxytryptamine (5‐HT) receptors (5‐HT_1A, 5‐HT₂ and 5‐HT₇) in the nucleus raphe magnus (NRM) on the ventilatory and thermoregulatory responses to hypoxia. Methods: To this end, pulmonary ventilation (V_E) and body temperature (T_b) of male Wistar rats were measured in conscious rats, before and after a 0.1 μL microinjection of WAY‐100635 (5‐HT_1A receptor antagonist, 3 μg 0.1μL^?1, 56 mm ), ketanserin (5‐HT₂ receptor antagonist, 2 μg 0.1μL^?1, 36 mm ) and SB269970 (5‐HT₇ receptor antagonist, 4 μg 0.1 μL^?1, 103 mm ) into the NRM, followed by 60 min of severe hypoxia exposure (7% O₂). Results: Intra‐NMR microinjection of vehicle (control rats) or 5‐HT antagonists did not affect V_E or T_b during normoxic conditions. Exposure of rats to 7% O₂ evoked a typical hypoxia‐induced anapyrexia after vehicle microinjections, which was not affected by microinjection of WAY‐100635, SB269970 or ketanserin. The hypoxia‐induced hyperpnoea was not affected by SB269970 and ketanserin intra‐NMR. However, the treatment with WAY‐100635 intra‐NRM attenuated the hypoxia‐induced hyperpnoea. Conclusion: These data suggest that 5‐HT acting on 5‐HT_1A receptors in the NRM increases the hypoxic ventilatory response.     

Mast Cells and 5-HT: Uptake of labelled 5-hydroxytryptamine (5-HT) and 5-hydroxytryptophan in relation to storage of 5-HT in individual rat mast cells

The capacity of individual rat peritoneal mast cells to take up and concentrate radioactively labelled 5-hydroxytryptamine (5-HT) and 5-hydroxytryptophan (5-HTP) was studied by quantitative cyto-chemical methods. By means of consecutive application of microfluorometric, autoradiographic and microinterferometric methods, the following parameters could be determined for each cell: 1. Relative amount of endogenous 5-HT. 2. Concentration of accumulated isotopes. 3. Dry mass. 4. Concentration of endogenous 5-HT. 5. Total amount of accumulated isotopes. The calculated correlations of these variables to each other showed general heterogeneity of the population, variations between the different cells being very large. However, on the whole, increasing dry mass of the mast cells was accompanied by an increase in 5-HT content, but with a decrease in concentrations of endogenous 5-HT and accumulated radioactive 5-HTP. Similarly, cells having large stores of endogenous 5-HT showed a higher total uptake, but a lower cellular concentration of exogenous 5-HT and 5-HTP, than cells that were poor in endogenous 5-HT. The results indicated that the uptake of ³H-5-HTP was proportional to the total area of the cell membrane, while the degree of 3-HT uptake was determined by other factors. When the concentrations of radioactivity in the incubation medium was compared to that of the living mast cell, it was found that the averagr cell was able to concentrate exogenous 5-HTP and 5-HT around 4 and 30 times, respectively.