Synaptic regulation of paraventricular arginine vasopressin-containing neurons by neuropeptide Y-containing monoaminergic neurons in rats

Summary Synaptic regulation of arginine vasopressin (AVP)-containing neurons by neuropeptide Y (NPY)-containing monoaminergic neurons was demonstrated in the paraventricular nucleus of the rat hypothalamus. NPY and AVP were immunolabeled in the pre- and the post-embedding procedures, respectively, and monoaminergic fibers were marked by incorporating 5-hydroxydopamine (5-OHDA), a false neurotransmitter. The immunoreaction for NPY was expressed by diaminobenzidine (DAB) chromogen, and that for AVP by gold particles. The DAB chromogen was localized on the surface of the membrane structures, such as vesicles or mitochondria, and on the core of large cored vesicles. Gold particles were located on the core of the secretory granules within the AVP cell bodies and processes. The incorporated 5-OHDA was found as dense cores within small or large vesicular structures. From these data, three types of nerve terminals were discernible: NPY-containing monoaminergic, NPY-containing non-aminergic, and monoaminergic fibers. The AVP cell bodies appeared to have synaptic junctions formed by these nerve terminals as well as by the unlabeled nerve terminals which have small clear vesicles and large cored vesicles. These different types of nerve terminals were frequently observed in a closely apposed position on the same AVP cell bodies. The functional relationships of these three types of neuronal terminals are discussed.     

Mapping,quantitative distribution and origin of substance P- and VIP-containing nerves in the Uvea of guinea pig eye

Summary VIP- and substance P-like immunoreactivities were found in considerable concentrations (VIP: 17.3±4.8 pmol/g, mean ± SEM; substance P:11.1±1.8 pmol/g) in the uveal portion of the guinea pig eye.d Immunocytochemistry localised these two regulatory peptides to nerve fibres found principally in a plexus in the iris (substance P) and in an extensive network surrounding the blood vessels of the choroid (VIP). A remarkable anatomical demarcation of the two types of peptide-containing nerves was established by the staining of substance P-containing nerves, which stops at the level of the ciliary body. This uveal area is known to be involved in the ocular responses to nociceptive stimuli. At the ultrastructural level, immunoreactivity for both peptides was localised to distinct subpopulations of p-type nerves, distinguishable by the size of their large dense-cored vesicles. Those immunoreactive for VIP were significantly larger (p<0.0005) than those immunoreactive for substance P (95±7 nm and 82±9 nm respectively; mean ± SD). Interruption of the trigeminal pathway produced a remarkable decrease of substance P immunoreactivity in the anterior portion of the uvea (9.1±1.5 pmol/g, mean ± SEM, control; 5.3±1.3 pmol/g, denervated), but not of VIP immunoreactivity in the choroid. Following colchicine treatment, VIP-immunoreactive neuronal cell bodies were localised in the choroid. The separate anatomical localisations and distributions of the two uveal peptides appear to be related to their different origins and functional roles in the response of the eye to noxious stimuli.To whom offprint requests should be sent     

烫伤对大鼠下丘脑视上核、室旁核AVP神经元的影响──免疫电镜观察及形态计量分析

为研究加压素与应激的关系,我们曾应用免疫细胞化学（ＰＡＰ）方法结合图像分析技术观察到烫伤应激后２小时视上核、室旁核加压素神经元免疫反应阳性物质的总体积减小最明显。本文采用免疫电镜技术,进一步观察了烫伤后２小时视上核、室旁核加压素神经元超微结构的变化,并运用体视学方法定量分析了对照组及烫伤后２小时组视上核、室分校加压素神经元各２４０张照片的粗面内质网、高尔基复合体及线粒体的体积密度（Ｖｖ）、面积密度（Ｓｖ）、面数密度（Ｎａ）等。结果显示：烫伤后２小时组视上核、室旁核加压素神经元的粗面内质网的Ｖｖ．Ｓｖ显著增大,说明合成功能已开始增强,但是,高尔基复合体的Ｖｖ、Ｓｖ及阳性分泌颗粒却显著减少,因而引起总体积的缩小。从超微结构水平进一步证实加压素参与应激过程。     

人胎儿下丘脑视上区生长抑素神经元发生学的研究

本研究采用了免疫细胞化学方法,首次系统观察了２２例９—２７周引产人胎儿下丘脑视上区的视上核和视交叉上核内的生长抑素免疫反应神经元（Ｓｏｍａｔｏｓｔａｔｉｎｉｍｍｕｎｏｒｅａｃｔｉｖｅｎｅｕｒｏｎ,简称ＳＯＭ－ＩＲ神经元）的发生、发育的形态学特征。从１６周开始,在视上核（ＳｕｐｒａｏｐｔｉｃａｌｎｕｃｌｅｕｓＳＯＮ）和交叉上核（ＳｕｐｒａｃｈｉａｓｍａｔｉｅｎｕｃｌｅｕｓＳＯＮ）内可观察到ＳＯＭ－ＩＲ神经元,在ＳＯＮ、ＳＯＭ－ＩＲ神经元数随着胎龄增加而增多,到２２周时数量最多,以后随着胎龄增加ＳＯＭ－ＩＲ神经元数出现逐渐减少现象;ＳＣＮ内的ＳＯＭ－ＩＲ神经元在１８周和２２周时出现两次高峰,研究结果提示,ＳＯＮ和ＳＣＮ内的ＳＯＭ－ＩＲ神经元在胚胎发育过程中出现消长现象,表明了ＳＯＭ－ＩＲ神经元在ＳＯＮ和ＳＣＮ内各有其独特的发育过程。     

Quantitative autoradiographic light- and electron microscopic studies on the retinohypothalamic connections in the rat

Summary Light microscopic autoradiography performed subsequent to intraocular injection of ³H-leucine revealed silver grains (SG) above axons of the optic tract which could be followed into the ventral and caudal portion of the suprachiasmatic nuclei (SCN) and above the contralateral anterior hypothalamic nucleus (AHN). By high resolution photometric measurement and computer processing the labelled areas were analysed, thus yielding statistical data of the relative grain distribution. The highest SG density was found in the ventrolateral part of both SCN (SCvl), confirming earlier reports concerning retinohypothalamic connections. That area exhibiting a cytoarchitecture different from the remaining nucleus was traversed, however, by numerous labelled axons. In the caudal part of both SCN a specific projection field of retinal fibres could be located. Here, almost no traversing fibres contribute to the rather circumscribed marked area. In the ventral part of the contralateral AHN, diffuse labelling well above background levels could be observed. Distinction between bypassing and terminating fibres within the SCvl could not be made using light microscopy. Analysis of SG distribution of the SCvl with electron microscopic autoradiography revealed a specific localization of SG within presynaptic terminals containing clear vesicles and pale mitochondria.     

Oxytocin, but not arginine vasopressin is involving in the antinociceptive role of hypothalamic supraoptic nucleus

Our pervious study has demonstrated that the hypothalamic supraoptic nucleus (SON) plays a role in pain modulation. Oxytocin (OXT) and arginine vasopressin (AVP) are the important hormones synthesized and secreted by the SON. The experiment was designed to investigate which hormone was relating with the antinociceptive role of the SON in the rat. The results showed that (1) microinjection of l-glutamate sodium into the SON increased OXT and AVP concentrations in the SON perfusion liquid, (2) pain stimulation induces OXT, but not AVP release in the SON, and (3) intraventricular injection (pre-treatment) with OXT antiserum could inhibit the pain threshold increase induced by SON injection of l-glutamate sodium, but administration of AVP antiserum did not influence the antinociceptive role of SON stimulation. The data suggested that the antinociceptive role of the SON relates to OXT rather than AVP.     

磁场对丘脑下部一氧化氮含量的影响及与丘脑下部神经内分泌核团的关系

应用硝酸还原酶反应—分光光度法测定和NADPH-d组织化学技术,对磁场处理后丘脑下部一氧化氮量的变化及其可能的原因进行了研究,发现磁场可促使丘脑下部一氧化氮量(OD值)显著升高,并具有显著滞后效应。NADPH-d阳性神经细胞及NADPH-d和血管加压素(AVP)双染阳性神经细胞集中分布在丘脑下部室旁核、室周核和视上核,但不存在 于视交叉上核,提示室旁核、室周核和视上核一氧化氮能神经细胞是丘脑下部的一氧化氮的主要来源。磁场处理后大鼠丘脑下部一氧化氮含量(OD值)较正常对照组显著升高应归因于这些神经细胞受磁场作用表达增强。一氧化氮和血管加压素的共存可能对磁场调节内分泌具有一定意义。     

磁场对丘脑下部一氧化氮含量的影响及与丘脑下部神经内分泌核 …

By employing nitric acid reductase-spectrophotometry and NADPH-diaphorase/AVP cytochemistry technique, the effects of magnetic field on NO in hypothalamus and relations to Paraventricular Nucleus (PVN), Periventricular Nucleus (PEN), Supraoptic Nucleus (SON) and Suprachiasmatic Nucleus (SCN) were investigated. It was found that the NADPH-d positive neurons and some NADPH-d/AVP dually stained neurons existed in PVN, PEN, SON, but not in SCN, and the magnetic field induced NO (OD) increase there and the high NO (OD) level lasted for 3 hours. The results suggested that NO (OD) increase after the treatment of magnetic field in hypothalamus may result from strong expression of NOergic neurons in the PVN, PEN and SON. The coexistance of NO and AVP may play important role in the regulation of endocrine and neuroendocrine by the magnetic field.     

Effects of Morris water maze testing on the neuroendocrine stress response and intrahypothalamic release of vasopressin and oxytocin in the rat

Adult male Wistar rats were trained in the Morris water maze (MWM) on 3 consecutive days to find a visible platform. Concomitantly, microdialysis samples from the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei were collected in order to monitor local release of the neuropeptides vasopressin (AVP) and oxytocin (OXT), respectively, during controllable swim stress. Additionally, a separate set of animals was equipped with chronic jugular venous catheters to collect blood samples for analyzing plasma concentrations of corticotropin (ACTH) and corticosterone during training in the MWM. As measured by microdialysis, swimming in the MWM caused a significantly increased release of AVP within the PVN and of OXT within the SON on each of the 3 test sessions. In contrast to OXT in the SON, basal AVP concentrations in the PVN tended to rise from day to day. Plasma ACTH and corticosterone were found to be similarly elevated in response to MWM exposure on each of the test sessions. Taken together, these data demonstrate that testing in the MWM is not only associated with a significant activation of the hypothalamo-pituitary-adrenal axis but also with an intrahypothalamic release of AVP and OXT. If compared with findings using repeated forced swimming as an uncontrollable stressor (Wotjak, C.T., Ganster, J., Kohl, G., Holsboer, F., Landgraf, R., Engelmann, M., 1998. Dissociated central and peripheral release of vasopressin, but not oxytocin, in response to repeated swim stress: new insights into the secretory capacities of peptidergic neurons. Neuroscience 85, 1209-1222), the present results suggest that (1) similarities in the release profiles of AVP in the PVN and plasma hormone levels are fairly independent from the controllability of the stressor and seem, thus, to primarily relate to the physical demands of the task, whereas (2) the different intra-SON OXT release profiles might be linked to the controllability of the stressor.     

Morphological changes in cultured myotubes treated with agents that interfere with lysosomal function

Summary Treatment of cultured muscle cells with the inhibitors of lysosomal function, leupeptin, and chloroquine, decrease the degradation of acetylcholine receptors (AChR) and causes accumulation of undegraded receptors intracellularly. Under these conditions the number of cytoplasmic coated vesicles, i.e. structures that appear to transport this receptor within the cultured muscle cell, increases in parallel. This study investigates the effects of leupeptin and chloroquine on the morphology of cultured myotubes in order to learn more about the turnover of acetylcholine (ACh) receptors and the origin of the coated vesicles. Chloroquine causes involution of the plasma membrane, disorganization in the arrangement of sarcomeres, vacuolization, and enlargement of dense lysosome-like bodies in myotubes. The diameter of dense bodies in untreated myotubes is 0.36 ± 0.01 m (mean ± SEM) compared with 2 ± 0.12 m after 48 h of incubation with chloroquine. Leupeptin does not disrupt the normal architecture of sarcomeres and does not cause vacuolization of the myotubes. However, leupeptin does enlarge the dense bodies, although to a lesser extent than chloroquine (average diameter after 48 h treatment, 1.0 ± 0.06 m, p < 0.01). Untreated myotubes appear to contain equal numbers of large and small coated vesicles. After chloroquine treatment 95% of coated vesicles are large (80–120 nm in diameter), whereas after leupeptin treatment the majority of coated vesicles are small (40–70 nm in diameter). After incubation with horseradish peroxidase (HRP) 62% ± 9 of coated vesicles in chloroquinetreated cells contain the tracer, whereas in control cells only 11% ± 4 of coated vesicles contain HRP reaction product. These observations indicate that chloroquine causes accumulation of coated vesicles and interferes with degradation of AChR by preventing fusion of lysosomes with coated vesicles originating by endocytosis.     

Ultrastructure of the corpus cardiacum and corpus allatum of the house cricket Acheta domesticus

Summary The ultrastructure of the corpus cardiacum (CC) and corpus allatum (CA) of the house cricket, Acheta domesticus, is described. Axon profiles within the CC contain neurosecretory granules 160–350 nm in diameter which are indistinguishable from those found in type I neurosecretory cells of the pars intercerebralis and in the nervus corporis cardiaci I. The CC itself contains two cell types: intrinsic neurosecretory cells and glial cells. Intrinsic NSC cytoplasm contains Golgi bodies and electron dense neurosecretory granules 160–350 nm in diameter. Synaptoid configurations with 20–50 nm diameter electron lucent vesicles were observed within axon profiles of the CC. The structure of the CA is relatively uniform with one cell type predominating. Typical CA cells possess large nucleoli, active Golgi complexes, numerous mitochondria, and occassional microtubules. Groups of dark staining cells scattered throughout the CA of some animals were interpreted as evidence of cellular death.This work was done while JTB was supported by USPHS Training Grant HD-0266 from NICHDI wish to express my thanks to Dr. Richard A. Cloney for sharing his expertise in electron microscopy     

Reactive oxygen species are physiological mediators of the noradrenergic signaling pathway in the mouse supraoptic nucleus

Free radicals are essential for the vasopressin (AVP) response to plasmatic hyperosmolarity. Noradrenergic afferents are the major projections on the supraoptic nucleus (SON) of the hypothalamus and stimulate the expression of AVP via a nitric oxide (NO) pathway. In this study, we investigated the mechanisms linking free radicals and noradrenaline (NA)-induced regulation of AVP. Analysis of Tg8 transgenic mice, invalidated for the monoamine oxidase-A gene and with consequently high levels of brain monoamines and AVP in the SON, showed that free radicals are more abundant in their SON than in that of wild-type mice (WT). Antioxidant superoxide dismutase 1 and 2 and catalase enzyme activities were also higher in these mice than in WT. This may explain the observed absence of cytotoxicity that would otherwise be associated with such high level of free radicals. Treatment of Tg8 mice with α-MPT, a blocking agent for NA synthesis, decreased both the production of free radicals and the AVP levels in the SON. Furthermore, incubation of ex vivo slices including the SON with NA increased the production of free radicals and AVP levels in wild-type mice. When NA was associated with α-lipoic acid, an antioxidant blocking the production of free radicals, AVP remained at its control level, indicating that free radicals are required for the effect of NA on the expression of AVP. In slices incubated with SNP, a producer of NO, free radicals and AVP levels increased. When NA was associated with L-NAME (a NO synthase blocker), the levels of free radicals and AVP were the same as in controls. Thus, the noradrenaline–NO pathway, which stimulates the expression of vasopressin, involves free radicals. This study provides further evidence of the physiological importance of free radicals, which should no longer be considered solely as cytotoxic factors.     

中国树鼩下丘脑视上核和室旁核内加压素和催产素能神经元的比较分布

应用PAP-PAAP双重免疫组化染色程序在同一切片上进行两种肽能物质的定位,观察了中国树鼩下丘脑视上核和室旁核内VP能和OT能神经元的比较解剖学分布,发现:视上核被视束分成主部和交叉后部。在视上核主部,其头侧部几乎仅含OT能神经元胞体,中间部VP能胞体出现并逐渐增多,尾侧部VP能胞体数目明显超过OT能胞体。在明显含有两种胞体的中间部和尾侧部,OT能胞体多位于背内侧,VP能胞体多位于腹外侧;在视上核交叉后部,其头侧部以VP能胞体为主,且多位于背外侧,OT能胞体多位于腹内侧。中间部OT能胞体多位于内侧,VP能胞体多位于外侧。尾侧部OT能胞体多位于背、腹两侧,VP能胞体则多位于中间;在室旁核,其头侧部几乎全由OT能胞体构成。中间部,VP能胞体出现并逐渐增多,OT和VP能胞体分别主要位于内、外侧。尾侧部两种神经元胞体较明显地分为内、外两群,内侧群主要为OT能胞体,外侧群几乎全为VP能胞体,该群的头侧半又可分为背腹两个亚群,至尾侧半,此二亚群渐合并。本文讨论了OT和VP能神经元在中国树鼩和大鼠视上核和室旁核内的比较分布。     

Locus Coeruleus Lesions Decrease Oxytocin and Vasopressin Release Induced by Hemorrhage

The role of the noradrenergic nucleus Locus Coeruleus (LC) on hemorrhage-induced vasopressin (AVP) and oxytocin (OT) secretion was examined. Rats with LC lesion were submitted to three 1-min hemorrhage sessions at 5-min intervals; 15% of the total blood volume was withdrawn in each session. OT and AVP were measured in plasma, paraventricular (PVN) and supraoptic (SON) nuclei and in posterior pituitary (PP). LC Lesion did not affect basal plasma AVP or OT levels, but partly blocked the increase in plasma AVP and OT induced by hemorrhage. Hemorrhage produced decreases in content of AVP and OT in the PVN and SON and increased levels in the PP. These responses were attenuated in the lesioned group, but only in the PVN and PP. Data suggest a stimulatory role of the inputs from LC to PVN neurons on hemorrhage-induced OT and AVP secretion and that, this pathway is critical in the hypo-volemic neuroendocrine reflex.Special Issue Dedicated to Miklós Palkovits.     

The Notch effector gene Hes1 regulates migration of hypothalamic neurons, neuropeptide content and axon targeting to the pituitary

Proper development of the hypothalamic-pituitary axis requires precise neuronal signaling to establish a network that regulates homeostasis. The developing hypothalamus and pituitary utilize similar signaling pathways for differentiation in embryonic development. The Notch signaling effector gene Hes1 is present in the developing hypothalamus and pituitary and is required for proper formation of the pituitary, which contains axons of arginine vasopressin (AVP) neurons from the hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON). We hypothesized that Hes1 is necessary for the generation, placement and projection of AVP neurons. We found that Hes1 null mice show no significant difference in cell proliferation or death in the developing diencephalon at embryonic day 10.5 (e10.5) or e11.5. By e16.5, AVP cell bodies are formed in the SON and PVN, but are abnormally placed, suggesting that Hes1 may be necessary for the migration of AVP neurons. GAD67 immunoreactivity is ectopically expressed in Hes1 null mice, which may contribute to cell body misplacement. Additionally, at e18.5 Hes1 null mice show continued misplacement of AVP cell bodies in the PVN and SON and additionally exhibit abnormal axonal projection. Using mass spectrometry to characterize peptide content, we found that Hes1 null pituitaries have aberrant somatostatin (SS) peptide, which correlates with abnormal SS cells in the pituitary and misplaced SS axon tracts at e18.5. Our results indicate that Notch signaling facilitates the migration and guidance of hypothalamic neurons, as well as neuropeptide content.     

Development of the human hypothalamus

The hypothalamus has been claimed to be involved in a great number of physiological functions in development, such as sexual differentiation (gender, sexual orientation) and birth, as well as in various developmental disorders including mental retardation, sudden infant death syndrome (SIDS), Kallman's syndrome and Prader-Willi syndrome. In this review a number of hypothalamic nuclei have therefore been discussed with respect to their development in health and disease. The suprachiasmatic nucleus (SCN) is the clock of the brain and shows circadian, and seasonal fluctuations in vasopressin-expressing cell numbers. The SCN also seems to be involved in reproduction, adding interest to the sex differences in shape of the vasopressin-containing SCN subnucleus and in its VIP cell number. In addition, differences in relation to sexual orientation can be seen in this perspective. The vasopressin and VIP, neurons of the SCN develop mainly postnatally, but as premature children may have circadian temperature rhythms, a different SCN cell type is probably more mature at birth.Thesexually dimorphic nucleus (SDN, intermediate nucleus, INAH-1), is twice as large in young male adults as in young females. At the moment of birth only 20% of the SDN cell number is present. From birth until two to four years of age cell numbers increase equally rapidly in both sexes. After this age cell numbers start to decrease in girls, creating the sex difference. The size of the SDN does not show any relationship to sexual orientation in men. The large neurosecretory cells of thesupraoptic (SON) andparaventricular nucleus (PVN) project to the neurohypophysis, where they release vasopressin and oxytocin into the blood circulation. In the fetus these hormones play an active role in the birth process. Fetal oxytocin may initiate or accelerate the course of labor. Fetal vasopressin plays a role in the adaptation to stress—caused by the birth process—by redistribution of the fetal blood flow.Corticotropin-releasing hormone (CRH) neurons of the PVN play a central role in stress response. Thus fetal CRH neurons may play a role in the timing of the moment of birth. Recently, alterations have been described in peptidergic, aminergic and cholinergic transmitters in the hypothalamus in SIDS. Future research will have to establish whether these changes are part of the course of SIDS. A large proportion of the SON and PVN neurons also produce tyrosine hydroxylase (TH). In neonates the majority of TH-immunoreactive neurons colocalizes vasopressin, while in the adult the majority of TH-positive neurons colocalizes oxytocin. TH-expression might be a sign of hyperactivation, for example from perinatal hypoxia.Oxytocin neurons also project to the brain stem. These neurons have an inhibitory effect on eating. Interestingly, in the Prader-Willi syndrome, characterized for example by insatiable hunger, we have found that the number of oxytocin-expressing neurons is about half the normal value. It can be concluded that the various hypothalamic nuclei are involved in a great number of functions and show clear and differential changes in development with respect to sexual differentiation, birth and a number of diseases. I believe that only a small proportion of such changes has at present been revealed.Special issue dedicated to Dr. Robert Balázs     

中缝大核在刺激视上核镇痛中的作用

运用核团灌流液的放免测定和高压液相色谱法以及核团内注射拮抗剂,观察了化学刺激下丘脑视上核（ＳＯＮ）对中缝大核（ＮＲＭ）灌流液内催产素（ＯＴ）、精氨酸加压素（ＡＶＰ）和５－羟色胺（５－ＨＴ）含量的影响以及ＮＲＭ内注射ＡＶＰ、５－ＨＴ或ＯＴ受体拮抗剂对痛阈（ＰＴ）的影响。结果表明：ＳＯＮ内注射Ｌ－谷氨酸（Ｌ－Ｇｌｕ）１０μｇ后动物痛阈明显升高,ＮＲＭ灌流液中ＯＴ和５－ＨＴ的含量明显高于对照组水平,ＡＶＰ的含量仅有一过性增加。ＮＲＭ内注射ｏＴ或５－ＨＴ拮抗剂可逆转化学刺激ＳＯＮ引起的镇痛作用;而ＡＶＰ的Ｖ＿(１／２)受体拮抗剂也轻度抑制这种镇痛作用,但Ｖ＿１拮抗剂对此作用无影响。以上结果提示：在刺激ＳＯＮ镇痛中,ＯＴ起着重要作用,Ｌ－Ｇｌｕ刺激ＳＯＮ的ＯＴ细胞释放ＯＴ,作用于ＮＲＭ细胞的ＯＴ受体和Ｖ＿２受体而产生镇痛作用,５－ＨＴ在此过程中也发挥重要作用。     

Histochemical and ultrastructural study on the innervation of the byssus glands of Mytilus galloprovincialis

The aluminium-formaldehyde (ALFA) histofluorescence method reveals an extensive plexus of brilliant greenish monoaminergic elements in the glandular zones of the Mytilus foot, while only scanty nerve fibres are acetylcholinesterase-positive. By electron microscopy, bundles of nerve fibres can be seen i) in close connection with the intrinsic musculature located in the connective septa among the glands, and ii) near the cell bodies and necks of all the byssus glands. The nerve fibres show varicosities containing three types of vesicles: small clear (50-60 nm), small granular (80-90 nm), and large granular (160-200 nm). The regions of close apposition between nerve terminals and muscle or gland cells generally do not show typical pre- or postsynaptic specializations. Along the pedal groove, mainly in the proximal two thirds of the foot, peripheral bipolar neurons can be detected, both by fluorescence and electron microscopy.     

Substance P in the suprachiasmatic nucleus of the rat: an immunohistochemical and in situ hybridization study

Using a biotin-streptavidin-horseradish peroxidase (HRP) immunohistochemical technique the distribution of substance P-immunoreactive neuronal elements was investigated in the rat suprachiasmatic nucleus (SCN). Substance P-immunoreactive nerve fibres and varicosities were distributed throughout the suprachiasmatic nucleus, with the largest accumulation in its ventral part. Because this location overlaps with the innervation of retinal afferents, the distribution and density of substance P-immunoreactive fibres in bilaterally enucleated rats were compared to normal rats. The density of substance P-immunoreactive fibres and nerve terminals in the ventral part of the suprachiasmatic nuclei was reduced in the rats with bilateral destruction of the optic nerves, whereas the density of fibres and nerve terminals in the dorsal part as well as other retinal target areas in the thalamus and mesencephalon was unaffected. In rats pretreated with an intraventricular injection of colchicine several substance P-immunoreactive perikarya were identified in the suprachiasmatic nucleus. The immunoreactive neurons, measuring 9.7 m±1.1 m in diameter, were frequently observed in the central core of the nucleus and to a lesser extent in the dorsomedial and ventrolateral subparts. Using in situ hybridization histochemistry pre-protachykinin-A mRNA was found in the same part of the SCN indicating that synthesis of substance P takes place in SCN neurons. Using a double immunohistochemical approach applying diaminobenzidine and benzidinedihydrochloride as chromagens substance P-, vasoactive intestinal peptide (VIP)-, and vasopressin/neurophysin-immunoreactivities were identified in the same brain section. The substance P-immunoreactive perikarya constituted a separate population of SCN neurons, which were not vasopressin-, neurophysin- or VIP-immunoreactive. Taken together, these observations show that substance P is contained in the retinohypothalamic pathway and within a group of SCN cell bodies, indiating that substance P may play a role in the generation and entrainment of circadian rhythmicity.     

Organotypic Suprachiasmatic Nuclei Cultures of Adult Voles Reflect Locomotor Behavior: Differences in Number of Vasopressin Cells

This study is the first to demonstrate organotypic culturing of adult suprachiasmatic nuclei (SCN). This approach was used to obtain organotypic SCN cultures from adult vole brain with a previously determined state of behavioral circadian rhythmicity. We examined vasopressin (AVP) immunoreactivity in these organotypic slice cultures. AVP is one of the major neuropeptides produced by the SCN, the main mammalian circadian pacemaker. AVP immunoreactivity in the SCN of adult common voles in vivo has been shown to correlate with the variability in expression of circadian wheel-running behavior. Here, cultures prepared from circadian rhythmic and nonrhythmic voles were processed immunocytochemically for AVP. Whereas in all cultures AVP could be observed, AVP immunoreactivity differed considerably between vole SCN cultures. SCN cultures from rhythmic voles contained significantly lower numbers of AVP immunoreactive (AVPir) cells per surface area than cultures from nonrhythmic voles. The correlation between timing of behavior and AVP immunoreactivity in vitro is similar to the correlation found earlier in vivo. Apparently, such correlation depends on intrinsic AVP regulation mechanisms of SCN tissue, and not on neural or hormonal input from the environment, as present in intact brain.