Ultrastructural demonstration of dopamine-beta-hydroxylase immunoreactive nerve terminals on vasopressin neurons in the paraventricular nucleus of the rat by double-labeling immunocytochemistry

The noradrenergic innervation of vasopressin (VP) neurons in the paraventricular nucleus (PVN) of the rat was studied ultrastructurally by double-labeling immunocytochemistry combining the preembedding peroxidase-antiperoxidase method for dopamine-beta-hydroxylase (DBH) with the post-embedding immunogold staining method for neurophysin II, the carrier protein of VP. DBH-like immunoreactive nerve terminals were found to make synaptic contacts with neurophysin II-like immunoreactive neuronal perikarya and their processes. This provides morphological evidence for noradrenergic control of the release of VP, at the PVN of the rat.     

大鼠下丘脑催产素样和加压素样神经元与5-羟色胺及儿茶酚胺能传入纤维末梢的关系

用免疫组织化学ABC双标记法,显示下丘脑各神经分泌核区中催产素样免疫反应(oxytocin-like immunoreactivity,OT-li)、加压素样免疫反应(vasopressin-like immunoreaetivity,VP-li神经元及5-羟色胺样免疫反应(serotonin-like immunoreactivity,5-HT-li)和儿茶酚胺能,即酪胺酸羟化酶免疫反应(tyrosine hydroxylase-like immunoractivity,TH-li)纤维末梢,对这两种单胺能纤维末梢在视上核、室旁核及各种神经分泌副核中的分布,及其与OT-li和VP-li神经元相关的特点及异同加以分析。结果显示,5-HT-li与TH-li纤维末梢在下丘脑视上核、室旁核及各神经分泌副核中,与OT-li及VP-li神经元相接触的多寡程度多不相同。视上核、室旁核内有极高密度的TH-li纤维末梢,与OT-li及VP-li神经元相接触,并在多数神经分泌副核的OT-li及Vp-li神经元周围,也有多寡不等的分布;而中等至高密度的5-HT-li纤维末梢,主要与各神经分泌副核内的OT-li或VP-li神经元相接触。说明5-HT-li和儿茶酚胺能传入纤维末梢,可能分别侧重调控不同神经分泌核区的大细胞神经分泌细胞群。     

The role of catecholaminergic neurons in the hypothalamus and medullary visceral zone in response to restraint water-immersion stress in rats

The activity of catecholaminergic neurons in the hypothalamus and the medullary visceral zone (MVZ) in rats in response to restraint water-immersion stress (RWIS) was measured by use of dual Fos and tyrosine hydroxylase (TH) immunohistochemistry. In RWIS rats Fos immunoreactive (Fos-IR) nuclei dramatically increased in the paraventricular nucleus (PVN), the supraoptic nucleus (SON), the dorsal motor nucleus of the vagus (DMV), the nucleus of the solitary tract (NTS), the area postrema (AP), and the ventrolateral medulla (VLM). A small number of TH-immunoreactive (TH-IR) and Fos/TH double-labeling neurons in the PVN, and their absence from the SON, were observed in both RWIS and nonstressed rats. More TH-IR neurons were observed in the MVZ of RWIS rats than in nonstressed rats. In RWIS and nonstressed rats, the percentage of Fos-IR nuclei in TH-IR neurons was 38.0 and 14.3% in the DMV, 34.4 and 9.7% in the NTS, 18.6 and 4.5% in the AP, and 45.7 and 18.9% in the VLM, respectively. In conclusion, catecholaminergic neurons in the MVZ are involved in the response to RWIS; although the PVN and SON also participate in the response to RWIS, the mechanism is not via catecholaminergic neurons.     

后叶加压素在下丘脑的分布和释放途径及失水对正中隆起中后叶加压素的影响

我们用免疫细胞化学方法(ICC)对大白鼠下丘脑中加压素(VP)免疫反应阳性的神经元作了较详细研究。观察到VP阳性神经元存在于下丘脑室旁核(PVN)各亚核、视上核(SON)、交叉上核(SCN)、室周核(PN)及一些附属核团,包括环状核(CN)、交叉后核(RCN)、下丘脑外侧核(HLN)、穹窿周围核(PFN)。且发现一特殊的VP阳性胞体聚集区—很可能是多巴胺神经元聚集区A_(14)中的细胞,位于第三脑室侧壁中1/3段两侧,腹内侧核背侧。本文首次观察到在PVN和SON之间有VP阳性的神经纤维相联系。ICC和免疫电镜研究进一步证明在正中隆起外带存在VP阳性纤维,含大颗粒囊泡的VP末梢紧邻门脉毛细血管。将HRP注入第四脑室用HRP逆行追踪与ICC方法相结合,在PVN中观察到双标细胞,与直接用ICC法所见VP阳性轴突伸入第三脑室腔的结果一致。说明PVN中存在接触脑脊液神经元。干渴动物正中隆起的内、外带中的VP免疫反应明显减弱。     

Comparative study of dopamine- and noradrenaline-immunoreactive terminals in the paraventricular and supraoptic nuclei of the rat

The distribution of dopaminergic and noradrenergic terminal fields of the paraventricular (PVN) and supraoptic (SON) nuclei of the rat was investigated at the optic and electron microscopical level using antibodies directed against dopamine (DA) and noradrenaline (NA). The DA innervation was uniform among these nuclei, although more important in the PVN than in the SON. NA fibers were preferentially distributed in the parvocellular parts of the PVN and in areas of the magnocellular nuclei where vasopressinergic neurons were mainly located. Both DA and NA terminals synaptically contacted magnocellular neurons on their cell body or dendrites. This study thus provides morphological evidence for a double and independent catecholaminergic control, by DA and NA, on neuroendocrine mechanisms at the hypothalamic level.     

腹腔注射2-脱氧-D-葡萄糖可诱发大鼠视上核和室旁核神经元表达Fos

目的 探讨腹腔注射2-脱氧-D-葡萄糖（2-DG）能否激活大鼠下丘脑视上核（SON）和室旁核（PVN）神经元而表达Fos。方法 健康雄性SD大鼠12只,随机分为腹腔注射2-DG组（6只）、生理盐水对照组（3只）及正常对照组（3只）。各自处理后,应用免疫组织化学方法,观察各组下丘脑SON和PVN内Fos表达及其与催产素（OT）和加压素（VP）的双标情况,同时采用ELISA方法对血清中OT和VP的含量进行检测。 结果与生理盐水对照组和正常对照组相比,2-DG引发的特异性Fos免疫阳性产物主要集中分布于下丘脑外侧区和穹隆周区,在SON、PVN也有密集表达。SON和PVN内的Fos表达与该区的特异性神经活性物质OT和VP有共存。OT/Fos双标细胞率（双标细胞占OT阳性细胞的百分率）在SON和PVN分别为87.10%、90.57%,明显高于VP/Fos在这两个核团的双标率（双标细胞占VP阳性细胞的百分率,68.42%、76.92%）,两者比较差异有统计学意义（P<0.05）。ELISA检测结果显示,2-DG组动物血清中OT和VP水平与对照组相比无明显变化。 结论 腹腔注射2-DG可激活大鼠下丘脑SON和PVN内OT和VP神经元表达Fos,SON和PVN可能参与2-DG诱导的急性应激反应。     

Monoclonal anti-vasopressin (VP) antibodies penetrate into VP neurons,in vivo

Summary The fate of monoclonal anti-vasopressin antibodies (VP-MAbs) injected in vivo into the paraventricular nucleus (PVN) of the rat brain was studied by immunocytochemistry. Depending on the post survival time, VP-MAbs contained in an ascites fluid were stained at different levels of the VP neurons: the cytoplasm of the PVN neurons, the fibres of the median eminence and the granular layer of the Gyrus Dentatus. The identification of endogenous peptides synthesized by PVN neurons showed that the VP-MAbs uptake was specific: it did not appear either in the oxytocinergic neurons or in the non immunoreactive neurons of the Brattleboro rat brain, this rat being genetically incapable of synthesizing central VP. Conversely, VP-MAbs only penetrated into the VP neurons: ascites fluid containing monoclonal antibodies prepared against bovine thyroglobulin (the carrier conjugated to VP in our immunizations) was neither stained in magnocellular neurons nor carried in nerve fibres. The neuronal uptake and transport of VP-MAbs occurred in vivo: they were totally inhibited by heating of the ascites fluid at 56° C for 30 min; this treatment did not alter the VP-MAbs themselves but probably destroyed some thermic sensitive component essential to the macromolecule internalization. The biological effects of antibodies injected in vivo have been reported. The results described here suggest that some specific antibodies passively transferred into the brain could act directly on the peptide synthesis recognized by the antibodies.This study was supported by grants from INSERM (CRE 84 60 03)     

加压素能和宫缩素能神经元在新生儿下丘脑的分布

本文用ABC法对加压素样和宫缩素样神经元在新生儿下丘脑的分布进行了观察。结果发现,两者的神经元集中分布于视上核、室旁核及副大细胞分泌核。根据两者的分布特点,又将视上核、室旁核和副大细胞分泌核再分为若干分区,各分区均以加压素样神经元为主,约占总数的80～90％。本文结果提示加压素样和宫缩素样神经元在下丘脑的分布具有动物种属和年龄特征。     

Noradrenergic innervation of vasopression-containing neurons in the rat hypothalamic supraoptic nucleus

The noradrenergic innervation of vasopressin (VP)-containing neurons in the supraoptic nucleus (SON) of the rat hypothalamus was studied electron microscopically by using double-labeling immunocytochemistry combining the pre-embedding peroxidase-anti-peroxidase method with post-embedding immunocolloidal gold staining. Noradrenaline-like immunoreactive axon terminals were found to make synaptic contacts with neurophysin II-like immunoreactive neurons in the SON. This study provides morphological evidence for noradrenergic control of neuronal activity of VP-containing neurons at the SON level.     

脑室注射组胺对下丘脑室旁核促皮质素释放激素神经元的作用

目的:观察第三脑室注射组胺对下丘脑室旁核促皮质素释放激素(CRH)神经元活动的影响。方法:Fos癌蛋白免疫组化LSAB法结合双抗原标记法;半定量逆转录聚合酶链反应(RT-PCR)方法。结果:第三脑室注射组胺后,(1)下丘脑室旁核Fos阳性神经元数目明显增加(P＜0.05);(2)室旁核内的Fos阳性神经元中约有31.78%同时呈CRH阳性反应;(3)室旁核CRHmRNA含量明显升高,且有量效关系。结论:中枢组胺可以激活下丘脑室旁核的CRH神经元,并使CRH基因表达增加。     

切除大鼠肾上腺对丘脑下部CRF、VP和OT的影响——免疫组织化学研究

几乎只有向脑室内注入秋水仙素之后,才能在其丘脑下部室旁核(PVN)显示促肾上腺皮质激素释放因子(CRF)免疫反应神经元。然而,我们切除肾上腺也能显示这类神经元,而且其细胞数目在术后3周时明显多于1周。切除肾上腺又用秋水仙素处理,细胞的数目及其免疫反应强度增加得更加显著。切除肾上腺1、3周后,PVN前份背内侧区出现一群淡染的VP样神经元。术后PVN内OT样神经元无明显变化。在正中隆起(ME)处:切除肾上腺1周时,外带CRF样纤维免疫反应强度较正常明显减弱,3周时又恢复到正常水平。1周时减弱的原因本文作了推测。外带VP样纤维的免疫反应在术后1周时不减弱,而是增加,3周时增加得更明显。OT样纤维束居于ME内带,术后1周时没有明显变化,3周时其免疫反应有所增强。切除肾上腺后即用地塞米松治疗,结果PVN及ME内CRF、VP免疫反应与未治疗者比较,均受到不同程度的抑制。上述结果证明,肾上腺对CRF和VP均具有反馈抑制作用,并说明它们都参与了丘脑下部-腺垂体-肾上腺轴的调控。然而,对OT还需进一步研究。     

Evidence for the medullary visceral zone as a neural station of neuroimmunomodulation

The aim of the present study was to test the possibility that the catecholaminergic projectional pathway from the vagus nerve to the medullary visceral zone (MVZ) thence to the paraventricular nucleus of hypothalamus (PVN) was involved in the cytokine-to-brain communication. A triple labeling method in which WGA-HRP retrograde tracing was combined with anti-Fos and -TH immunohistochemical staining was used. WGA-HRP was stereotaxically injected into unilateral PVN in the rat, after a survival of 48 h, animals received intraperitoneal injection of lipopolysaccharide (LPS). The distribution of the HRP retrogradely labeled neurons, Fos protein positive and catecholaminergic neurons (tyrosine hydroxylase as marker) in the MVZ was observed. Subdiaphragmatic vagotomy (SDV) and sham surgery were also used to observe the different Fos expression in the MVZ after intraperitoneal administration of lipopolysaccharide (LPS) or pyrogen-free saline (NS). Under light microscope, seven types of positively stained neurons could be distinguished within the MVZ, namely neurons single-labeled with Fos, HRP or TH, respectively; neurons double-labeled with Fos/TH, Fos/HRP or HRP/TH separately; and neurons triple-labeled with Fos, HRP and TH staining. Intraperitoneal LPS caused lots of robust Fos expression within the MVZ in the sham surgery groups and this response in the MVZ was markedly inhibited in the vagotomized rats. The results suggested that some catecholaminergic neurons in the MVZ could send projections to the PVN and this pathway might be involved in the relay of peripheral immune information via vagus nerve. MVZ was a neural relay station in the immune-to-brain communication and might play a significant role in the neuroimmunomodulation via vagus-MVZ-PVN pathway.     

大鼠下丘脑室旁核神经元的超微结构特征

在透射电镜下,我们观察到PVN大细胞具有四种不同的形态,认为属于同一类细胞分泌活性的不同时相:(1)合成相,粗面内质网非常丰富,(2)加工相:Golgi复合体发达,周围分布着一些神经分泌颗粒;(3)储存相:神经分泌颗粒明显增多:(4)转运相:细胞质内神经分泌颗粒很少,而在细胞周围可见一些充满神经分泌颗粒的突起。免疫电镜的结果也证实了VP阳性神经细胞具有类似的四种不同形态,其中在合成相,粗面内质网囊膜呈免疫阳性反应,还有一些PAP免疫复合物沉积在细胞质中。而在加工,储存和转运相,神经分泌颗粒呈免疫阳性反应。同样,小细胞亦具有三种不同形态特证,可能代表着不同的功能状态。     

大鼠延髓至下丘脑室旁核的儿茶酚胺能投射神经元在胃伤害性刺激后的FOS表达

用ＨＲＰ注入下丘脑室旁核逆行追踪与抗Ｆｏｓ和抗酪氨酸羟化酶（ＴＨ）双重免疫细胞化学相结合的三重标记方法，对大鼠孤束核和延髓腹外侧区至下丘脑室旁核的儿茶酚胺能投射神经元对胃伤害性刺激后的ｃ－ｆｏｓ表达进行了观察，发现孤束核和延髓腹外侧区有７种不同的标记细胞：ＨＲＰ、Ｆｏｓ、ＴＨ单标细胞，Ｆｏｓ／ＨＲＰ、Ｆｏｓ／ＴＨ、ＨＲＰ／ＴＨ双标细胞，Ｆｏｓ／ＨＲＰ／ＴＨ三标细胞。上述７种标记细胞主要分布在延髓中、尾段孤束核的内侧亚核、连合亚核和延髓腹外侧区以及两者之间的网状结构。ＨＲＰ标记细胞以注射侧为主，对侧有少量分布。本文结果证明，大鼠孤束核和延髓腹外侧区至下丘脑室旁核投射的部分儿茶酚胺能神经元可能参与胃伤害性刺激的传导和调控。     

延髓内脏带儿茶酚胺能神经元对LPS免疫刺激的Fos表达

目的　探讨“延髓内脏带 (MVZ)至下丘脑室旁核 (PVN)”的儿茶酚胺能通路在“免疫 脑通讯”中的作用。　方法　应用WGA HRP逆行追踪与抗Fos和抗酪氨酸羟化酶 (TH)抗体双重免疫组织化学相结合的三重标记方法 ,即先将WGA HRP注入大鼠一侧PVN内 ,存活 4 8h后经腹腔注射免疫刺激剂脂多糖 (LPS)诱发免疫反应后 ,观察MVZ中WGA HRP逆标细胞、Fos蛋白和儿茶酚胺能神经元 (以TH为标记物 )的分布及表达状况。　结果　在MVZ内发现 7种阳性神经元 ,即HRP、Fos、TH单标细胞、Fos HRP、Fos TH、HRP TH双标细胞和Fos HRP TH三标细胞。Fos HRP双标记和Fos HRP TH三标记细胞分别占总HRP逆行标记细胞的 12 5 %和 39 6 %。　结论　MVZ对LPS免疫刺激起反应 ,并可能将免疫信息经儿茶酚胺能神经元上传至PVN ;MVZ可能作为“免疫 脑通讯”的一个中继站 ,通过“MVZ→PVN”通路起免疫调节作用。     

含后叶加压素的视上核神经元及含P物质的中缝背核神经元至下丘脑室旁核投射的研究

用HRP逆行追踪与免疫细胞化学结合的方法,对某些投射至大鼠下丘脑室旁核的神经元的化学性质进行了研究.结果显示在视上核内存在三种标记细胞:HRP单标细胞、后叶加压素免疫反应阳性单标细胞和HRP后叶加压素双标细胞.双标细胞为大、中型椭圆形或圆形细胞,约占HRP标记细胞总数的22%.在中缝背核投射至室旁核的神经元中,部分为P物质免疫反应阳性,双标细胞为中小型梭形细胞,约占HRP标记细胞总数的20%.上述结果提示:视上核有后叶加压素能神经元、中缝背核有P物质能神经元投射到室旁核.     

Vasopressin increases GABAergic inhibition of rat hypothalamic paraventricular nucleus neurons in vitro

This investigation used an in vitro hypothalamic brain slice preparation and whole cell and perforated-patch recording to examine the response of magnocellular neurons in hypothalamic paraventricular nucleus (PVN) to bath applications of vasopressin (VP; 100-500 nM). In 22/38 cells, responses were characterized by an increase in the frequency of bicuculline-sensitive inhibitory postsynaptic potentials or currents with no detectable influence on excitatory postsynaptic events. Perforated-patch recordings confirmed that VP did not have an effect on intrinsic membrane properties of magnocellular PVN neurons (n = 17). Analysis of intrinsic membrane properties obtained with perforated-patch recording (n = 23) demonstrated that all of nine VP-sensitive neurons showed a rebound depolarization after transient membrane hyperpolarization from rest. By contrast, 12/14 nonresponding neurons displayed a delayed return to resting membrane potentials. Recordings of reversed inhibitory postsynaptic currents with chloride-loaded electrodes showed that responses to VP persisted in media containing glutamate receptor antagonists but were abolished in the presence of tetrodotoxin. In addition, responses were mimicked by vasotocin [Phe(2), Orn(8)], a selective V(1a) receptor agonist, and blocked by [beta-Mercapto-beta, beta-cyclopentamethylenepropionyl(1),O-Me-Tyr(2), Arg(8)]-VP (Manning compound), a V(1a)/OT receptor antagonist. Neither [deamino-Cys(1),Val(4),D-Arg(8)]-VP, a selective V(2) receptor agonist, nor oxytocin were effective. Collectively, the results imply that VP acts at V(1a) receptors to excite GABAergic neurons that are presynaptic to a population of magnocellular PVN neurons the identity of which features a unique rebound depolarization. Endogenous sources of VP may be VP-synthesizing neurons in suprachiasmatic nucleus, known to project toward the perinuclear regions of PVN, and/or the magnocellular neurons within PVN.     

GABA regulates corticotropin releasing hormone levels in the paraventricular nucleus of the hypothalamus in newborn mice

The paraventricular nucleus of the hypothalamus (PVN) is a major regulator of stress responses via release of corticotropin releasing hormone (CRH) to the pituitary gland. Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is characteristic of individuals with major depressive disorder (MDD). Postmortem data from individuals diagnosed with MDD show increased levels of CRH mRNA and CRH immunoreactive neurons in the PVN. In the current study, an immunohistochemical (IHC) analysis revealed increased levels of CRH in the PVN of newborn mice lacking functional GABA_B receptors. There was no difference in the total number of CRH immunoreactive cells. By contrast, there was a significant increase in the amount of CRH immunoreactivity per cell. Interestingly, this increase in CRH levels in the GABA_B receptor R1 subunit knockout was limited to the rostral PVN. While GABAergic regulation of the HPA axis has been previously reported in adult animals, this study provides evidence of region-specific GABA modulation of immunoreactive CRH in newborns.     

下丘脑室旁核经迷走神经复合体对哮喘大鼠的神经调控研究

为探讨下丘脑室旁核对哮喘大鼠的神经调控途径,取健康雄性SD大鼠制备哮喘模型并诱发哮喘发作,运用WGA-HRP逆行追踪法与免疫组织化学染色法(ABC法)相结合的双重标记方法,在光镜下观察向迷走神经复合体(DVC)发出投射的下丘脑室旁核(PVN)神经元内Fos蛋白的表达情况。结果显示:PVN内有三种阳性细胞,即HRP逆行标记神经元、Fos样免疫阳性神经元和HRP/Fos双标神经元。这些神经元主要见于PVN小细胞部的内侧亚核和外侧亚核,散在分布于背侧亚核,在前小细胞亚核内未见阳性反应;Fos样免疫阳性细胞呈双侧分布,且HRP逆标神经元,HRP/Fos双标神经元也为双侧分布,但以注射区同侧占优势。HRP/Fos双标神经元占HRP标记细胞的44.22%。本研究结果提示,哮喘大鼠发作时中枢内包括下丘脑PVN、延髓DVC的多个脑区内神经元兴奋,且两者之间通过直接投射联系参与哮喘的调控。     

Involvement of the septum in the regulation of paraventricular vasopressin neurons by the subfornical organ in the rat

Extracellular recordings were obtained from 58 phasically active neurosecretory neurons in the hypothalamic paraventricular nucleus (PVN) of urethane-anesthetized male rats. Of these PVN neurons, 39 exhibited an increase and 11 displayed a reduction in ongoing activity following electrical stimulation of the subfornical organ (SFO), while the remaining neurons were unresponsive. Microinjection of the local anesthetic lidocaine into the medial septum reversibly abolished the SFO stimulus-evoked reduction in 7 out of 9 PVN neurons tested, whereas similar injection was without effect on the stimulus-evoked increase in 18 out of 20 PVN neurons tested. These results suggest that the SFO efferents through the medial septum to the PVN exert a predominantly inhibitory influence on the excitability of putative vasopressin (VP)-secreting neurons in the PVN.