Synaptic association between enkephalin-containing axon terminals and proopiomelanocortin-containing neurons in the arcuate nucleus of rat hypothalamus

By employing an electron microscopic dual immunolabeling technique, a synaptic association between neurons containing immunoreactive adrenocorticotropin (ACTH) and axonal terminals containing immunoreactive methionine-enkephalin octapeptide (Enk-8) was found in the arcuate nucleus of the rat hypothalamus. The axonal terminals contained many small clear vesicles and some large cored vesicles. At the synaptic portions, membrane specialization was asymmetric.     

Estradiol target sites immunoreactive for beta-endorphin in the arcuate nucleus of rat and mouse hypothalamus

Combined autoradiography and immunocytochemistry was performed to examine estradiol-concentrating neurons of the arcuate nucleus in the hypothalamus of ovariectomized rats and mice for beta-endorphin-like immunoreactivity. A high number of [3H]estradiol-labeled cells occurred in the arcuate nucleus of both species. Neurons immunoreactive for beta-endorphin could be visualized throughout the arcuate nucleus as well as ventrolaterally outside of this nucleus. Colocalization of [3H] estradiol nuclear labeling and beta-endorphin-like cytoplasmic immunoreactivity could be found scattered throughout the arcuate nucleus, most frequently in the ventromedial part, although most of the [3H]estradiol target neurons did not react with the antibodies. The results suggest that a subpopulation of beta-endorphin neurons is directly addressed by estradiol and that the arcuate nucleus contains different groups of estradiol receptive neurosecretory cells.     

下丘脑弓状核KNDy神经元参与围绝经期潮热发生的机制

潮热是围绝经期女性最常见的特异性症状,严重危害女性的身心健康和生活质量。由于潮热的发病机制尚未明确,且现有的雌激素替代疗法存在诸多局限和禁忌,故探讨潮热的发病机制并寻找新的治疗靶点尤为迫切和重要。近年研究提示,围绝经期雌激素降低时下丘脑弓状核（ARC）中KNDy神经元异常是引起潮热的关键因素。部分学者认为,ARC中KNDy神经元通过调控促性腺激素释放激素及其下游促黄体生成素的脉冲释放参与潮热的发生;而另有学者认为,ARC中KNDy神经元通过调控下丘脑视前区的正中视前核在潮热发生过程中发挥关键作用。我们就上述两种关于ARC KNDy神经元与潮热发生之间的关系及其参与潮热的可能机制进行总结和概括,为探寻诊治围绝经期潮热的新靶点和新方法奠定理论基础。     

Three-dimensional visualization of the distribution of melanin-concentrating hormone producing neurons in the mouse hypothalamus

We present here a new procedure to represent the 3D distribution of neuronal cell bodies within the brain, using exclusively softwares free for research purposes.Our technique is based on digitalized photos of brain slices processed by immunohistochemical technique, and the 3D Slicer software. The technique presented enables transposition of immunohistochemical or in situ hybridization data to the stereotaxic mouse brain atlas (e.g. Paxinos, G., Franklin, K.B.J., 2001. The Mouse Brain in Stereotaxic Coordinates. second ed. Academic Press, San Diego). By exporting the finalized models into a popular 3D design software (3DS Max) arbitrary environment and motion simulation can be created to improve the visual understanding of the area studied.Application of this technique provides the possibility to store, analyze and compare data – e.g. on the hypothalamic neuropeptides – across experimental techniques and laboratories.The method is exemplified by visualizing the distribution of immunohistochemically identified melanin-concetrating hormone (MCH) containing perikarya within the mouse hypothalamus.     

Orexin A activates leptin-responsive neurons in the arcuate nucleus

Orexins, also named hypocretins, are newly described neuropeptides, which are produced almost exclusively in neurons of the lateral hypothalamus and have been shown to increase food intake after intracerebroventricular injection. Leptin, the ob-gene product released from white adipocytes, is suspected to reduce food intake mainly by acting on neurons in the arcuate nucleus of the hypothalamus. Application of orexin A activated 85% (66 out of 78) of all neurons of the rat arcuate nucleus investigated electrophysiologically in an in vitro slice preparation, by a direct excitatory postsynaptic effect. Leptin inhibited electrical activity in 10 out of 22 orexin-sensitive neurons in this brain region and excited only 3 neurons. These data give the first indication as to where and how orexin might interact with the leptin-responsive hypothalamic network.     

Regionalized differentiation of CRH,TRH, and GHRH peptidergic neurons in the mouse hypothalamus

According to the updated prosomeric model, the hypothalamus is subdivided rostrocaudally into terminal and peduncular parts, and dorsoventrally into alar, basal, and floor longitudinal zones. In this context, we examined the ontogeny of peptidergic cell populations expressing Crh, Trh, and Ghrh mRNAs in the mouse hypothalamus, comparing their distribution relative to the major progenitor domains characterized by molecular markers such as Otp, Sim1, Dlx5, Arx, Gsh1, and Nkx2.1. All three neuronal types originate mainly in the peduncular paraventricular domain and less importantly at the terminal paraventricular domain; both are characteristic alar Otp/Sim1-positive areas. Trh and Ghrh cells appeared specifically at the ventral subdomain of the cited areas after E10.5. Additional Ghrh cells emerged separately at the tuberal arcuate area, characterized by Nkx2.1 expression. Crh-positive cells emerged instead in the central part of the peduncular paraventricular domain at E13.5 and remained there. In contrast, as development progresses (E13.5–E18.5) many alar Ghrh and Trh cells translocate into the alar subparaventricular area, and often also into underlying basal neighborhoods expressing Nkx2.1 and/or Dlx5, such as the tuberal and retrotuberal areas, becoming partly or totally depleted at the original birth sites. Our data correlate a topologic map of molecularly defined hypothalamic progenitor areas with three types of specific neurons, each with restricted spatial origins and differential migratory behavior during prenatal hypothalamic development. The study may be useful for detailed causal analysis of the respective differential specification mechanisms. The postulated migrations also contribute to our understanding of adult hypothalamic complexity.     

Cardiodilatin-immunoreactive neurons in the hypothalamus of Tupaia

Summary Using various region specific antibodies raised against partial sequences of synthetic cardiodilatin (CDD) we detected immunoreactive neurons with their perikarya in the nucleus periventricularis of Tupaia belangeri. The fibers could be traced laterally directed towards the amygdaloid complex and some varicosities were also observed in the lateral parts of the nucleus periventricularis. It is postulated that brain CDD represents a new neuropeptide and that these CDD-IR neurons are involved in specific functions related to the modulation of the cardiovascular centers.     

Interferon modulates glucose-sensitive neurons in the hypothalamus

 Interferon-α (IFN) therapy induces feeding suppression that resembles anorexia. The hypothalamic glucose-sensitive neurons engage in feeding behavior. Coronal sections of rat brains, containing both the lateral hypothalamus (LH) and the ventromedial hypothalamus (VMH), as well as single-cell recordings were used to study the interaction between IFN and glucose-sensitive neurons. IFN suppressed the majority (78%) of LH neurons, while reduction in glucose concentration elicited excitation in the majority (85%) of the same neurons. The opposite effects were observed in the VMH, where IFN excited the majority of neurons (61%), and reduction in glucose concentration exerted the opposite effects in 64% of VMH recordings. Concomitant IFN and glucose reduction exhibited only the effects elicited by IFN, regardless of whether the glucose reduction caused excitation (LH) or suppression (VMH). This observation suggests that IFN causes anorexia by modulating the LH and VMH glucose-sensitive neurons. Received: 6 August 1996 / Accepted: 4 February 1997     

Dopamine and alpha-endorphin are contained in different neurons of the arcuate nucleus of hypothalamus as revealed by combined fluorescence histochemistry and immunohistochemistry

An attempt was made to demonstrate dopamine and alpha-endorphin neurons in the same sections of the arcuate nucleus by combining the techniques of fluorescence histochemistry and immunohistochemistry. Dopamine neurons were found mainly in the medial part near the wall of the third ventricle and alpha-endorphin neurons in the lateral part. We failed to detect any neurons showing simultaneous dopamine fluorescence and alpha-endorphin positive immunoreactivity. We conclude therefore that dopamine synthesizing neurons are probably different from alpha-endorphin synthesizing neurons in the arcuate nucleus.     

Reaction of neurons of arcuate region of hypothalamus to microiontophoretic application of serotonin at different stages of the estrous cycle in rats

Sensitivity to serotonin was investigated in single neurons of the arcuate region of the hypothalamus as the center of tonic regulation of gonadotropic function in conditions of its microiontophoretic application in different stages of the estrous cycle in rats. It was shown that at the diestrus-II stage of the estrous cycle, especially during morning hours, cells predominate among neurons of the arcuate region with activated reaction to serotonin. During proestrus increased representation of cells with inhibited reaction is noted among spontaneously functioning neurons, which is especially pronounced during evening hours of this stage of the cycle. Serotonin induced reduction in the level of luteinizing hormone in blood and this effect was usually combined with an activating effect of indolamine on functioning of neurons. The combination of a delayed effect of the neurotransmitter on spontaneous activity of neurons of the arcuate nucleus with a reduced level of luteinizing hormone in blood was noted in the overwhelming majority of cases only at the proestrus stage.Translated from Fiziologicheskii Zhurnal SSSR imeni I. M. Sechenova, Vol. 69, No. 7, pp. 881–886, July, 1983.     

Peripheral ghrelin selectively increases Fos expression in neuropeptide Y - synthesizing neurons in mouse hypothalamic arcuate nucleus

Ghrelin, a peptide isolated from the rat stomach, is the endogenous ligand of the growth hormone-secretagogue receptor and also known to have orexigenic effect. We examined the influence of intraperitoneal (i.p.) injection of ghrelin on food intake and brain neuronal activity in freely fed mice. Ghrelin (3, 10 or 30 microg/mouse) dose-dependently increased food intake by 0.8-, 1.6- and 2.6-fold, respectively, at 30 min post injection. Ghrelin (10 microg/mouse) induces Fos expression selectively in the ventromedial part of the hypothalamic arcuate nucleus (Arc). No change in Fos expression was observed in other hypothalamic and hindbrain nuclei. About 90% of the Fos-positive neurons in the Arc expressed neuropeptide Y (NPY) messenger RNA. These data indicate that NPY neurons in the Arc are likely the primary target mediating i.p. ghrelin induced orexigenic effect.     

Triiodothyronine enhances the morphological maturation of dopaminergic neurons from fetal mouse hypothalamus cultured in serum-free medium

In dissociated hypothalamic cell cultures of 16-day mouse embryos, growing in chemically defined medium, the catecholaminergic neurons were identified by autoradiography after labelling with [3H]dopamine and by immunocytochemistry with an anti-tyrosine hydroxylase antibody. Using selective inhibitors of amine transport and radioenzymatic determination of amine levels in these cultures, we show that these neurons were mostly dopaminergic. The number of dopaminergic neurons identified by the two techniques increased between days 5 and 8 and decreased after 15 days in vitro. The same number of neurons were identified by autoradiography and by immunocytochemistry and consisted of fusiform and multipolar neurons. The proportion of both types remained steady until 15 days in vitro. Under these conditions, the addition of triiodothyronine (10(-9) M) at the initiation of the culture increased the size but not the number of dopaminergic neurons after 8 days in vitro. Furthermore, triiodothyronine significantly increased the dopaminergic neurite length and arborization. This morphological effect of triiodothyronine was associated with an increase of 35% in [3H]dopamine uptake. Our study shows that hypothalamic dopaminergic neurons are responsive to triiodothyronine which acts as a maintenance or trophic factor having an effect on neurite extension and arborization.     

P2X receptors are expressed on neurons containing luteinizing hormone-releasing hormone in the mouse hypothalamus

Expression of P2X₁, P2X₂, P2X₃, P2X₄, P2X₅ and P2X₆ receptors, members of a family of ATP-gated cation channels, on neurons containing luteinizing hormone-releasing hormone (LHRH) in the mouse hypothalamus was studied with double-labeling fluorescence immunohistochemistry. This study demonstrated that different combinations of P2X receptor subunits were expressed on the perikarya and axon terminals of LHRH-producing neurons. It was shown for the first time that P2X₂, P2X₄, P2X₅ and P2X₆ receptor subunits were expressed on the perikarya of LHRH-producing neurons and P2X₂ and P2X₆ on their axon terminals. These results suggest that activation of P2X receptors by ATP via different homomeric or heteromeric P2X receptors at both presynaptic and postsynaptic sites could be involved in the regulation of LHRH secretion at the forebrain level.     

Glucose-sensitive neurons in the rat arcuate nucleus contain neuropeptide Y.

Glucose is known to regulate the activity of the hypothalamic feeding centers. Neuropeptide Y (NPY)-containing neurons in the hypothalamic arcuate nucleus (ARC) have been implicated in the stimulation of feeding. We examined the presence of glucose-sensitive neurons in the ARC and their coincidence with NPY-containing neurons. Cytosolic Ca2+ concentration ([Ca2+]i) in single ARC neurons isolated from rat hypothalamus was measured with fura-2 fluorescence imaging; the cells were then stained immunocytochemically with an anti-NPY antiserum. Lowering the glucose concentration from 10 to 1 mM increased [Ca2+]i in 36 out of 180 neurons (20%), the majority of which (34 neurons, 94%) were immunoreactive for NPY. In conclusion, the ARC contains glucose-sensitive NPY-containing neurons. The suggested role of these neurons is to transduce a reduction in the glucose concentration in the brain to the release of NPY and, subsequently, stimulation of feeding.     

Orexigen-sensitive NPY/AgRP pacemaker neurons in the hypothalamic arcuate nucleus

The hypothalamic arcuate nucleus (ARC) integrates and responds to satiety and hunger signals and forms the origins of the central neural response to perturbations in energy balance. Here we show that rat ARC neurons containing neuropeptide Y (NPY) and agouti-related protein (AgRP), which are conditional pacemakers, are activated by orexigens and inhibited by the anorexigen leptin. We propose a neuron-specific signaling mechanism through which central and peripheral signals engage the central neural anabolic drive.     

Serotonin innervation of neuropeptide Y-containing neurons in the rat arcuate nucleus

Structural non-synaptic appositions between serotonin (5-HT) nerve endings and neuropeptide Y (NPY)-containing neurons were demonstrated in the rat arcuate nucleus by means of a combined radioautographic and immunocytochemical detection of [3H]5-HT uptake sites and NPY-immunoreactivity. Such cellular relationships are proposed to constitute a morphological substrate for putative 5-HT/NPY interactions in neuroendocrine hypothalamus.