Characterization of galanin-like immunoreactivity in the rat brain: effects of neonatal glutamate treatment

Concentrations of the neuropeptide, galanin, were measured using a newly characterized radioimmunoassay in brain regions of adult male rats treated neonatally with monosodium glutamate. Galanin-like immunoreactivity (galanin-IR) was significantly reduced 57% in the median eminence, 15% in the medial basal hypothalamus, and 27% in the septal region when compared to untreated littermates. Concentrations of galanin-IR were reduced 22% in the preoptic region and unchanged in the parietal cortex. These studies suggest that glutamate-sensitive, galanin-containing neurons in the arcuate nucleus project to regions of the basal forebrain of the rat in addition to the median eminence. The galanin projection from the arcuate nucleus to the median eminence suggests that this peptide plays a role in the regulation of anterior pituitary hormone secretion.     

The effect of arcuate nucleus transplantation on the development of the anterior pituitary in monosodium glutamate-treated rats

Treatment with monosodium glutamate (MSG) during the neonatal period is known to produce a selective lesion of the arcuate nucleus in rat brain, which is the major site of production of growth hormone releasing-hormone (GRH), followed by a secondary reduction in growth hormone (GH) synthesis in the anterior pituitary. Normal arcuate nuclei from hypothalamic areas of newborn rats were transplanted into the third ventricles of 27-day-old rats which were treated with MSG on alternate days for the first 10 days of life. Ninety days after birth, the anterior pituitaries were examined for GH synthesis by immunohistochemical staining with GH antiserum. The results indicated that the impaired GH synthesis in the anterior pituitary treated with MSG was partially restored in some recipients by grafts of arcuate nuclei in which the GRH-containing neurons were clearly detected by immunohistochemical staining with GRH antiserum.     

Activation of hypothalamic arcuate but not paraventricular neurons following carotid body chemoreceptor stimulation in the rat

The effect of carotid body chemoreceptor stimulation on 292 neurons in midline hypothalamic nuclei has been examined electrophysiologically in ethyl carbamate/sodium pentobarbitone anaesthetized rats. Experiments demonstrated that specific stimulation of carotid body chemoreceptors activates a small group (16) of neurons in the mediobasal hypothalamic arcuate nucleus, but has no effect on neurons (157) in the hypothalamic paraventricular nucleus or the anterior hypothalamus. Of 16 arcuate neurons activated by the stimulus, six projected to the median eminence and three projected to the dorsal medulla, as defined by antidromic invasion. Three of the neurons activated from the carotid body also showed a resting discharge that was linked with ventilation rate, suggesting that the arcuate nucleus may have some involvement with respiratory processing. The activation of neurons projecting to the median eminence implies that the release of adenohypophyseal hormones may also be influenced by carotid body chemoreceptors.     

Increase in pituitary dopaminergic receptors after monosodium glutamate treatment

We investigated whether a decrease in arcuate nucleus dopamine (DA) levels resulting from neonatal treatment with monosodium glutamate (MSG) affects the anterior pituitary DA receptors in adult male rats. MSG treatment resulted in a significant reduction in medial basal hypothalamic (MBH) DA levels, no change in its norepinephrine (NE) and epinephrine (E) concentrations, and a marked increase in circulating prolactin (PRL). Scatchard analyses of DA binding characteristics to anterior pituitary membranes using [3H]spiperone revealed linear plots, suggesting a single class of high-affinity, low-capacity binding sites. The DA binding capacity was significantly higher in MSG-treated rats than in controls with no change in affinity. The data indicate that anterior pituitary DA receptors change in accordance with altered physiological conditions. The increase in the number of DA receptors following destruction of the arcuate nucleus is probably a direct effect of reduced DA levels reaching the anterior pituitary gland.     

Specialized junction in the distal convoluted tubule of rat kidney

The effect of the monosodium glutamate (MSG) induced lesion of the arcuate nucleus on catecholamines in the arcuate nucleus and median eminence of the mouse hypothalamus was determined using the Falck-Hillarp histofluorescence technique. The number of fluorescent perikarya in the arcuate nucleus of treated animals was decreased approximately 60%; the fluorescence intensity of surviving neurons was notably reduced. These changes were accompanied by a reduction in the intensity of fluorescence in the median eminence. Pretreatment of control and MSG-lesioned animals with a monoamine oxidase inhibitor (pargyline) greatly increased fluorescence in the median eminence and arcuate nucleus of both groups. However, the number of fluorescing perikarya of the arcuate nucleus of the normal pargyline treated group far exceeded that of the pargyline MSG animals. It is concluded that neonatally administered MSG caused destruction of a large number of dopaminergic arcuate perikarya.     

Immunohistochemical evidence of neurons with GHRH or LHRH in the arcuate nucleus of male mice and their possible role in the postnatal development of adenohypophysial cells

Background: The neonatal administration of monosodium L-glutamate (MSG) has been used in investigations of the possible role of the arcuate nucleus in neuroendocrine regulation during postnatal development. We used this method to examine whether the mouse arcuate contained cell bodies immunoreactive with antisera to growth hormone releasing hormone (GHRH) or luteinizing hormone releasing hormone (LHRH), and whether these hypothalamic peptides affect synthesis and secretion of growth hormone and gonadotropin and the testis. Methods: The hypothalamus, pituitary, and testes of adult male mice treated with MSG during the neonatal period were fixed in Bouin's fluid or 10% neutral formalin. The hypothalamus was used in immune staining, the pituitary was used in both morphometry and immune staining, and the testis was stained with hematoxylin and eosin. Results: Body weights in control and treated mice were not different. The treated mice had more subcutaneous adipose tissue and a shorter body than the control mice. The testes were heavier in the controls. Many perikarya immunoreactive with antisera to GHRH or LHRH were found in the arcuate nucleus in control mice, but few such perikarya were found in this nucleus in treated mice. The size of the anterior lobe and the number and size of GH cells, follicle stimulating hormone (FSH) cells, and prolactin (PRL) cells in treated mice were less than those of control mice. Conclusions: GHRH and LHRH neurons in the arcuate nucleus in male mice may cause body and testis weight to increase via GH and LH cells, respectively, in the adenohypophysis during postnatal development. There are some differences in the hypothalamo-pituitary-testis axis of mice and rats. © 1994 Wiley-Liss, Inc.     

Neonatal monosodium glutamate dosing alters the sleep-wake cycle of the mature rat

Alterations of the sleep-wake cycle have been studied in male adult rats after neonatal administration of monosodium glutamate (MSG; 4 X 4 mg/g body wt.). Results indicated that MSG treatment caused: an almost complete disappearance of ACTH and alpha-MSH immunoreactive (IR) perikarya in the rostral part of the arcuate nucleus; an increase in total sleep duration with a more pronounced effect on paradoxical sleep. Regarding circadian rhythmicity there was a trend to a decomposition of the 24 h period into ultradian components (12 h, 8 h, 6 h harmonics). The participation of pro-opiomelanocortin peptides in sleep regulation is discussed.     

Changes in the cholecystokinin-synthetizing hypothalamic system during experimental diabetes mellitus in rats

The investigation was performed in 96 Wistar rats. Diabetes mellitus was induced by single injection of 50 mg/kg of streptozotocin. Cholecystokinin (CCK) synthesizing neurons were identified in hypothalamic structures using indirect immunofluorescence. In latent period of diabetes (2 wks) number of CCK--immunopositive neurons increases, especially in paraventricular and suprachiasmatic nuclei, while in ventrolateral subnucleus of arcuate nucleus and parvicellular subnucleus of paraventricular nucleus areas occupied by immunoreactive material in neurons and their CCK content are reduced. By the end of wk 5 of the disease increase in number of CCK immunopositive neurons was registered only in medial parvicellular subnucleus of paraventricular nucleus whereas in other structures their number was reduced. The administration of CCK to intact animals causes increase of insulin content in endocrinocytes of pancreatic islets, but does not affect the level of hypoglycemia. The administration of the peptide to animals with diabetes leads to destruction of pancreatic islets, decline in endocrinocyte number and insulin content and marked hypoglycemia. Thus, the data obtained indicate the significant role of hypothalamic peptidergic system and CCK in regulation of beta-endocrinocyte function.     

Forebrain projections of arcuate neurokinin B neurons demonstrated by anterograde tract-tracing and monosodium glutamate lesions in the rat

Neurokinin B (NKB) and kisspeptin receptor signaling are essential components of the reproductive axis. A population of neurons resides within the arcuate nucleus of the rat that expresses NKB, kisspeptin, dynorphin, NK3 receptors and estrogen receptor α (ERα). Here we investigate the projections of these neurons using NKB-immunocytochemistry as a marker. First, the loss of NKB-immunoreactive (ir) somata and fibers was characterized after ablation of the arcuate nucleus by neonatal injections of monosodium glutamate. Second, biotinylated dextran amine was injected into the arcuate nucleus and anterogradely labeled NKB-ir fibers were identified using dual-labeled immunofluorescence. Four major projection pathways are described: (1) local projections within the arcuate nucleus bilaterally, (2) projections to the median eminence including the lateral palisade zone, (3) projections to a periventricular pathway extending rostrally to multiple hypothalamic nuclei, the septal region and BNST and dorsally to the dorsomedial nucleus and (4) Projections to a ventral hypothalamic tract to the lateral hypothalamus and medial forebrain bundle. The diverse projections provide evidence that NKB/kisspeptin/dynorphin neurons could integrate the reproductive axis with multiple homeostatic, behavioral and neuroendocrine processes. Interestingly, anterograde tract-tracing revealed NKB-ir axons originating from arcuate neurons terminating on other NKB-ir somata within the arcuate nucleus. Combined with previous studies, these experiments reveal a bilateral interconnected network of sex-steroid responsive neurons in the arcuate nucleus of the rat that express NKB, kisspeptin, dynorphin, NK3 receptors and ERα and project to GnRH terminals in the median eminence. This circuitry provides a mechanism for bilateral synchronization of arcuate NKB/kisspeptin/dynorphin neurons to modulate the pulsatile secretion of GnRH.     

老年大鼠下丘脑视上核和弓状核的超微结构变化

观察老年大鼠下丘脑视上核腹侧大细胞神经元和弓状核神经元的超微结构,结果显示:老年大鼠视上核腹侧血管加压素(VP)神经元的胞体较成年鼠大,胞质结构比较致密,内含许多核糖体颗粒,粗面内质网分散成小泡状,神经分泌颗粒增多,溶酶体、线粒体、高尔基氏复合体数量都较多.而弓状核内的暗细胞和亮细胞的核糖体减少,高尔基氏复合体和线粒体都变化,还有少数神经元固缩,细胞质内胞器明显减少,呈现变性现象.结果提示:老年期下丘脑视上核VP神经元机能增强;而弓状核内两种细胞都显示机能减弱的征象.老年时下丘脑各核团神经元的这种不协调性变化,可能是动物衰老的一种重要表现.     

Cytometric investigations in histochemically identified neurons — Changes of fluorescence intensities and nuclear diameters in dopamine neurons of the arcuate nucleus in the cycling rat

Summary The relative formaldehyde-induced fluorescence intensities and the diameter of cell nuclei of fluorescent perikarya of the arcuate nucleus were recorded in serial cross-sectioned hypothalamic arcuate nuclei of regularly cycling rats (5 estrous and 4 proestrous animals). Stress-induced changes of cytometric parameters were avoided by preadaptation of animals to handling procedures. Dopamine neurons in a 75 m thick periventricular layer of the arcuate nucleus exhibited significantly smaller nerve cell nuclei and significantly reduced relative fluorescence intensities in proestrous rats. Both of these cytometric parameters indicate a decrease in the activity of periventricular dopamine neurons of the arcuate nucleus. The reported findings might support the hypothesis that dopamine inhibits the release of LH-RH.In partial fulfillment of requirements for an academic degree (Dr. med.)Supported by a grant from the Deutsche Forschungsgemeinschaft (-Ha 726/5-) and the Stiftung Volkswagenwerk (I/35 582) to H.G.H.     

Monosodium glutamate induced lesions of the arcuate nucleus. I. Endocrine deficiency and ultrastructure of the median eminence

Monosodium glutamate was administered daily on days 5 through 10 postnatally at a dose of 2.5 mg/gm body weight. Counts of remaining perikarya in the arcuate nucleus of adult mice indicated approximately an 80% decrease in the number of perikarya. The arcuate lesion resulted in endocrine deficits; reproductive capacity was reduced, animals were smaller in stature and obese, and the weights of the anterior pituitary, ovaries and testes were significantly decreased while adrenals were unaffected. Light microscopic studies revealed no significant changes in thickness or general histological appearance of the median eminence. At the electron microscope level, there were no alterations in the number of nerve terminals or dense core vesicles per unit area in the contact zone. These observations suggest that afferents to the median eminence from the arcuate nucleus may form a relatively small portion of its total nerve terminal population.     

Effects of monosodium glutamate on the development of intraventricular axons in the rat hypothalamus

The development of intraventricular axons in the infundibular recess of the young rat was investigated by correlative scanning and transmission electron microscopy (SEM-TEM). From the fourth through the fifteenth day of life such axons increase steadily in number. During subsequent weeks their number gradually decreases. In animals given monosodium glutamate on the fourth postnatal day there is widespread neuronal necrosis in the arcuate nucleus, and the development of intraventricular axons is greatly reduced. These findings suggest that the axons originate from the neurons of the arcuate nucleus.     

Synaptic regulation of proopiomelanocortin neurons can occur distal to the arcuate nucleus

Energy homeostasis is controlled to a large extent by various signals that are integrated in the hypothalamus. It is generally considered that neurons in each of the hypothalamic nuclei are regulated by afferent projections that terminate within the cell body region of the nucleus. However, here it is shown that hypothalamic proopiomelanocortin (POMC) neurons receive synaptic inputs onto distal dendrites that reside outside of the cell body region in the arcuate nucleus. Previous studies using whole cell recordings from identified neurons in brain slices have shown that cannabinoids reduce GABA release from inhibitory synapses onto the POMC cells. Here it was found that endocannabinoids inhibited GABAergic inhibitory postsynaptic currents in POMC neurons only in intact sagittal brain slices, but not coronal, horizontal, or sagittal slices that were truncated rostrally at the level of the optic chiasm. Thus endocannabinoids inhibited presynaptic GABA release only at an anatomically distinct subset of POMC-neuron dendrites that extends rostrally beyond the arcuate nucleus into preoptic hypothalamic regions. There are two key results. First, the activity of POMC neurons can be regulated by afferent input at sites much farther from the soma than previously recognized. Second, endocannabinoids can act to inhibit inputs only at selective dendrites. POMC neurons play a critical role in the maintenance of body weight. Therefore these data suggest that energy balance may be regulated, in part, by modulation of POMC neuron activity at sites outside of the arcuate nucleus.     

Non-Dopaminergic Neurons Expressing Dopamine Synthesis Enzymes: Differentiation and Functional Significance

The development and functional significance of neurons in the arcuate nucleus expressing tyrosine hydroxylase and/or aromatic L-amino acid decarboxylase were studied in rat fetuses, neonates, and adults using immunocytochemical (single and double immunolabeling of tyrosine hydroxylase and aromatic L-amino acid decarboxylase) methods with a confocal microscope and computerized image analysis, HPLC with electrochemical detection, and radioimmunological analysis. Single-enzyme neurons containing tyrosine hydroxylase were first seen on day 18 of embryonic development in the ventrolateral part of the arcuate nucleus. Neurons expressing only aromatic L-amino acid decarboxylase or both enzymes of the dopamine synthesis pathway were first seen on day 20 of embryonic development, in the dorsomedial part of the nucleus. On days 20–21 of embryonic development, dopaminergic (containing both enzymes) neurons amounted to less than 1% of all neurons expressing tyrosine hydroxylase and/or aromatic L-amino acid decarboxylase. Nonetheless, in the ex vivo arcuate nucleus and in primary neuron cultures from this structure, there were relatively high levels of dopamine and L-dihydroxyphenylalanine (L-DOPA), and these substances were secreted spontaneously and in response to stimulation. In addition, dopamine levels in the arcuate nucleus in fetuses were sufficient to support the inhibitory regulation of prolactin secretion by the hypophysis, which is typical of adult animals. During development, the proportion of dopaminergic neurons increased, reaching 38% in adult rats. Specialized contacts between single-enzyme tyrosine hydroxylase-containing and aromatic L-amino acid decarboxylase-containing neurons were present by day 21 of embryonic development; these were probably involved in transporting L-DOPA from the former neurons to the latter. It was also demonstrated that the axons of single-enzyme decarboxylase-containing neurons projected into the median eminence, supporting the secretion of dopamine into the hypophyseal portal circulation. Thus, dopamine is probably synthesized in the arcuate nucleus not only by dopaminergic neurons, but also by neurons expressing only tyrosine hydroxylase or aromatic L-amino acid decarboxylase.     

下丘脑移植的实验形态学研究

为了探讨脑组织移植的机制,本实验对大鼠进行了实验形态学研究。用谷氨酸单钠(MSG)损毁大鼠弓状核,用15～18天鼠胚下丘脑组织移入第三脑室,存活3～5个月。然后对正常(N)组,损毁(D)组及损毁后移植(T)组动物的下丘脑和垂体前叶进行光镜、电镜和免疫细胞化学观察,对血清的睾酮进行生化测定。结果表明:MSG能选择性地损毁弓状核内约80%的神经元。第三脑室内的移植物呈团块状,能存活并与宿主脑组织紧密连接,两者之间存在交界面或直接融合。荧光显微镜下可见移植物内有发黄绿色荧光的儿茶酚胺能神经细胞,免疫细胞化学显示移植区内存在TH阳性神经细胞,后者为梭形或多角形。电镜观察表明:移植区内有神经细胞、神经纤维、各种胶质细胞和毛细血管,并且存在轴-树和轴-轴突触。免疫电镜还显示有TH阳性神经元胞体和轴突末梢,后者可作为突触前成分与TH阴性结构形成突触。此外,免疫组化显示T组垂体前叶的GH和FSH(FSH在雄性动物能促进曲细精管发育和精子生成)细胞较D组明显增多,放免分析表明T组血清中睾酮含量也比D组明显升高。本研究还证明接触脑脊液的神经元在脑组织移植中有重要作用。     

Seasonal variations of the beta-endorphin neuronal system in the mediobasal hypothalamus of the jerboa (Jaculus orientalis)

The distribution of neurons expressing beta-endorphin immunoreactivity was explored in the brain of adult jerboa during two distinct periods characterizing its reproductive cycle. A large presence of cell bodies displaying beta-endorphin immunoreactivity occured within different parts of the mediobasal hypothalamus along its rostrocaudal extent, from the retrochiasmatic area to the posterior arcuate nucleus. Quantitatively, the highest density of immunoreactive beta-endorphin neurons was noted at the medial level of the arcuate nucleus. Furthermore, a seasonal study showed that the number of IR-beta-endorphin neurons was highest in the anterior portion of the arcuate nucleus of jerboas sacrificed in autumn as compared to those sacrificed during spring-summer. Quantitatively, the number of beta-endorphin containing neurons in autumn was 200% in comparison to that found in spring-summer. These results suggest that beta-endorphin containing neuronal population especially localized in the anterior part of arcuate nucleus, exerts an inhibitory influence on the GnRH neurosecretory system in the jerboa, notably in autumn, probably via an increasing expression of its products. The results provide morphofunctional arguments in favour of inhibitory opioid control of GnRH neurons activity and hence the neuroendocrine events regulating reproduction in jerboa.     

L-dopa-immunoreactive neurons in the rat hypothalamic tuberal region

The presence of L-DOPA-immunoreactivity is reported for the first time in the rat hypothalamic tuberal region. L-DOPA-immunoreactive neurons were demonstrated to be present in the ventrolateral part of the arcuate nucleus and periarcuate region just dorsal to the ventral surface of the brain (VLAR/PA). Weakly L-DOPA-immunostained neurons were found in the dorsomedial part of the arcuate nucleus and its neighboring periventricular nucleus (DMAR/PV). In contrast, dopamine (DA)-immunoreactive neurons were detected only in the DMAR/PV. These findings suggest that L-DOPA exists not only as a precursor of DA in neurons of the DMAR/PV, but also as an end-product in cells of the VLAR/PA.     

Thyrotropin releasing hormone (TRH): its widespread distribution in discrete hypothalamic nuclei and areas in rat brain

The precise distribution of thyrotropin releasing hormone (TRH) in 23 discrete brain nuclei and areas of Wistar strain male rats was determined by specific radioimmunoassay. TRH was detected in most of these areas. The highest concentration was found in the median eminence (27.52 +/- 2.84 ng/mg protein). The arcuate nucleus (4.92 +/- 0.58 ng/mg protein), dorsomedial nucleus (4.77 +/- 0.59 ng/mg protein) and medial preoptic area (3.94 +/- 0.15 ng/mg protein) also contained a considerable concentration of TRH. However, no TRH was detected in cerebral cortex, cerebellar hemisphere, anterior pituitary or pineal body. The data indicated that TRH was widely distributed throughout the hypothalamus; in particular, high concentrations occure in relatively restricted areas: in the median eminence, arcuate nucleus, dorsomedial nucleus and medial preoptic area. These areas coincide well with the so-called "thyrotropic" area of the hypothalamus.