Abstract
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1.
Melanin-concentrating hormone (MCH) and orexin-containing neurons participate in hypothalamic circuits that control energy homeostasis. While these two systems have projections to widespread target areas within the central nervous system, little is known about intrinsic characteristics and the molecular composition of both the MCH and orexin neurons themselves.
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By a combinatory approach of quantitative immunocytochemical identification and analysis with laser microdissection and semi-quantitative Real-time RT-PCR, here we present multi-transcriptional profiling of MCH and orexin neurons in the rat lateral hypothalamus.
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Immunocytochemical analysis showed that orexin peptide expression was increased after fasting both during the activity and resting period of rats, whereas MCH peptide content was only clearly upregulated at resting phase. Subsequent transcriptional profiling showed distinct expression patterns of MCH, orexin and cocaine-amphetamine regulated transcript (CART) between MCH and orexin neurons. A low expression level of dynorphin was found both in MCH and orexin neurons. Receptor expression profiles, reflecting interaction with neuropeptide Y, melanocortins, leptin, glucocorticoids and GABA, showed approximately similar expression patterns among the MCH and orexin neuronal systems. Expression of glutamate- and GABA-markers revealed a possible contributory role of both glutamate and GABA in functional output of MCH and orexin neurons.
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This method allowed differential screening at mRNA level after immunocytochemical neuron identification and analysis in heterogeneous brain regions, which can further specify functioning of the individual neurons. With respect to MCH and orexin neurons, this study emphasizes that these neurons are targets for stimulatory and inhibitory signals from other brain regions including the arcuate nucleus and the general circulation. Additionally, both glutamate and GABA appear to be involved in MCH and orexin neuronal functioning related to feeding and regulation of the energy balance.
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Abrahamson, E. E., Leak, R. K., and Moore, R. Y. (2001). The suprachiasmatic nucleus projects to posterior hypothalamic arousal systems. NeuroReport 12:435–440.
Antunes, V. R., Brailoiu, G. C., Kwok, E. H., Scruggs, P., and Dun, N. J. (2001). Orexins/hypocretins excite rat sympathetic preganglionic neurons in vivo and in vitro. Am. J. Physiol. Regul. Integr. Comp. Physiol. 281:1801–1807.
Bäckberg, M., Collin, M., Ovesjo, M. L., and Meister, B. (2003). Chemical coding of GABA B receptor-immunoreactive neurones in hypothalamic regions regulating body weight. J. Endocrinol. 15: 1–14.
Bäckberg, M., Ultenius, C., Fritschy, J. M., and Meister, B. (2004). Cellular localization of GABA receptor alpha subunit immunoreactivity in the rat hypothalamus: Relationship with neurones containing orexigenic or anorexigenic peptides. J. Endocrinol. 16:589–604.
Bahn, S., Augood, S. J., Ryan, M., Standaert, D. G., Starkey, M., and Emson, P. C. (2001). Gene expression profiling in the post-mortem human brain—No cause for dismay. J. Chem. Neuroanat. 22:79–94.
Bittencourt, J. C., Presse, F., Arias, C., Peto, C., Vaughan, J., Nahon, J. L., Vale, W., and Sawchenko, P. E. (1992). The melanin-concentrating hormone system of the rat brain: An immuno- and hybridization histochemical characterization. J. Comp. Neurol. 319:218–245.
Broberger, C., De Lecea, L., Sutcliffe, J. G., and Hökfelt, T. (1998). Hypocretin/orexin- and melanin-concentrating hormone-expressing cells form distinct populations in the rodent lateral hypothalamus: Relationship to the neuropeptide Y and agouti gene-related protein systems. J. Comp. Neurol. 402:460–474.
Broberger, C. (1999). Hypothalamic cocaine- and amphetamine-regulated transcript (CART) neurons: Histochemical relationship to thyrotropin-releasing hormone, melanin-concentrating hormone, orexin/hypocretin and neuropeptide Y. Brain Res. 848:101–113.
Bujs, R. M., Chun, S. J., Niijima, A., Romijn, H. J., and Nagai, K. (2001). Parasympathetic and sympathetic control of the pancreas: A role for the suprachiasmatic nucleus and other hypothalamic centers that are involved in the regulation of food intake. J. Comp. Neurol. 431:405–423.
Campbell, R. E., Smith, M. S., Allen, S. E., Grayson, B. E., Ffrench-Mullen, J. M., and Grove, K. L. (2003). Orexin neurons express a functional pancreatic polypeptide Y4 receptor. J. Neurosci. 23:1487–1497.
Castaño, J. P., Faught, W. J., Glave, E. E., Russell, B. S., and Frawley, L. S. (1997). Discordance of prolactin gene transcription, mRNA storage, and hormone release in individual mammotropes . Am. J. Physiol. 272:E390–E396.
Chemelli, R. M., Willie, J. T., Sinton, C. M., Elmquist, J. K., Scammell, T., Lee, C., Richardson, J. A., Williams, S. C., Xiong, Y., Kisanuki, Y., Fitch, T. E., Nakazato, M., Hammer, R. E., Saper, C. B., and Yanagisawa, M. (1999). Narcolepsy in orexin knockout mice: Molecular genetics of sleep regulation. Cell 98:437–451.
Chou, T. C., Lee, C. E., Lu, J., Elmquist, J. K., Hara, J., Willie, J. T., Beuckmann, C. T., Chemelli, R. M., Sakurai, T., Yanagisawa, M., Saper, C. B., and Scammell, T. E. (2001). Orexin (hypocretin) neurons contain dynorphin. J. Neurosci. 21:RC168.
Collin, M., Bäckberg, M., Ovesjo, M. L., Fisone, G., Edwards, R. H., Fujiyama, F., and Meister, B. M. (2003). Plasma membrane and vesicular glutamate transporter mRNAs/proteins in hypothalamic neurons that regulate body weight. Eur. J. Neurosci. 18:1265–1278.
Date, Y., Ueta, Y., Yamashita, H., Yamaguchi, H., Matsukura, S., Kangawa, K., Sakurai, T., Yanagisawa, M., and Nakazato, M. (1999). Orexins, orexigenic hypothalamic peptides, interact with autonomic, neuroendocrine and neuroregulatory systems. Proc. Natl. Acad. Sci. U.S.A. 96:748–753.
Drazen, D. L., Coolen, L. M., Strader, A. D., Wortman, M. D., Woods, S. C., and Seeley, R. J. (2004). Differential effects of adrenalectomy on melanin-concentrating hormone and orexin A. Endocrinology 145:3404–3412.
Elias, C. F., Saper, C. B., Maratos-Flier, E., Tritos, N. A., Lee, C., Kelly, J., Tatro, J. B., Hoffman, G. E., Ollmann, M. M., Barsh, G. S., Sakurai, T., Yanagisawa, M., and Elmquist, J. K. (1998). Chemically defined projections linking the mediobasal hypothalamus and the lateral hypothalamic area. J. Comp. Neurol. 402:442–459.
Emmert-Buck, M. R., Bonner, R. F., Smith, P. D., Chuaqui, R. F., Zhuang, Z., Goldstein, S. R., Weiss, R. A., and Liotta, L. A. (1996). Laser capture microdissection. Science 274:998–1001.
Fink, L., Kinfe, T., Stein, M. M., Ermert, L., Hanze, J., Kummer, W., Seeger, W., and Bohle, R. M. (2000). Immunostaining and laser-assisted cell picking for mRNA analysis. Lab. Invest. 80:327–333.
Goncharuk, V. D., Van Heerikhuize, J., Dai, J. P., Swaab, D. F., and Buijs, R. M. (2001). Neuropeptide changes in the suprachiasmatic nucleus in primary hypertension indicate functional impairment of the biological clock. J. Comp. Neurol. 431:320–330.
Hagan, J. J., Leslie, R. A., Patel, S., Evans, M. L., Wattam, T. A., Holmes, S., Benham, C. D., Taylor, S. G., Routledge, C., Hemmati, P., Munton, R. P., Ashmeade, T. E., Shah, A. S., Hatcher, J. P., Hatcher, P. D., Jones, D. N., Smith, M. I., Piper, D. C., Hunter, A. J., Porter, R. A., and Upton, N. (1999). Orexin A activates locus coeruleus cell firing and increases arousal in the rat. Proc. Natl. Acad. Sci. U.S.A. 96:10911–10916.
Hökansson, M. L., Brown, H., Ghilardi, N., Skoda, R. C., and Meister, B. (1998). Leptin receptor immunoreactivity in chemically defined target neurons of the hypothalamus. J. Neurosci. 18:559–572.
Hökansson, M., De Lecea, L., Sutcliffe, J. G., Yanagisawa, M., and Meister, B. (1999). Leptin receptor- and STAT3-immunoreactivities in hypocretin/orexin neurones of the lateral hypothalamus. J. Neuroendocrinol. 11:653–663.
Harthoorn, L. F., Oudejans, R. C., Diederen, J. H., Van de Wijngaart, D. J., and Van der Horst, D. J. (2001). Absence of coupling between release and biosynthesis of peptide hormones in insect neuroendocrine cells. Eur. J. Cell. Biol. 80:451–457.
Hill, J., Duckworth, M., Murdock, P., Rennie, G., Sabido-David, C., Ames, R. S., Szekeres, P., Wilson, S., Bergsma, D. J., Gloger, I. S., Levy, D. S., Chambers, J. K., and Muir, A. I. (2001). Molecular cloning and functional characterization of MCH2, a novel human MCH receptor. J. Biol. Chem. 276:20125–20129.
Horvath, T. L., Peyron, C., Diano, S., Ivanov, A., Aston-Jones, G., Kilduff, T. S., and Van den Pol, A. N. (1999). Hypocretin (orexin) activation and synaptic innervation of the locus coeruleus noradrenergic system. J. Comp. Neurol. 415:145–159.
Kamphuis, W., Schneemann, A., Van Beek, L. M., Smit, A. B., Hoyng, P. F. J., and Koya, E. (2001). Prostanoid receptor gene expression profile in human trabecular meshwork: A quantitative real-time PCR approach. Invest. Ophthalmol. Vis. Sci. 42:3209–3215.
Kiyashchenko, L. I., Mileykovskiy, B. Y., Maidment, N., Lam, H. A., Wu, M. F., John, J., Peever, J., and Siegel, J. M. (2002). Release of hypocretin (orexin) during waking and sleep states. J. Neurosci. 22:5282–5286.
Kristensen, P., Judge, M. E., Thim, L., Ribel, U., Christjansen, K. N., Wulff, B. S., Clausen, J. T., Jensen, P. B., Madsen, O. D., Vrang, N., Larsen, P. J., and Hastrup, S. (1998). Hypothalamic CART is a new anorectic peptide regulated by leptin. Nature 393:72–76.
Meijerink, J., Mandigers, C., Van de Locht, L., Tonnissen, E., Goodsaid, F., and Raemaekers, J. (2001). A novel method to compensate for different amplification efficiencies between patient DNA samples in quantitative real-time PCR. J. Mol. Diagn. 3:55–61.
Peyron, C., Tighe, D. K., Van den Pol, A. N., De Lecea, L., Heller, H. C., Sutcliffe, J. G., and Kilduff, T. S. (1998). Neurons containing hypocretin (orexin) project to multiple neuronal systems. J. Neurosci. 18:9996–10015.
Qu, D., Ludwig, D. S., Gammeltoft, S., Piper, M., Pelleymounter, M. A., Cullen, M. J., Mathes, W. F., Przypek, R., Kanarek, R., and Maratos-Flier, E. (1996). A role for melanin concentrating hormone in the central regulation of feeding behaviour. Nature 380:243–247.
Ramakers, C., Ruijter, J. M., Deprez, R. H., and Moorman, A. F. (2003). Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci. Lett. 339:62–66.
Rosin, D. L., Weston, M. C., Sevigny, C. P., Stornetta, R. L., and Guyenet, P. G. (2003). Hypothalamic orexin (hypocretin) neurons express vesicular glutamate transporters VGLUT1 or VGLUT2. J. Comp. Neurol. 465:593–603.
Saito, Y., Cheng, M., Leslie, F. M., and Civelli, O. (2001). Expression of the melanin concentrating hormone (MCH) receptor mRNA in the rat brain. J. Comp. Neurol. 435:26–40.
Sakurai, T., Amemiya, A., Ishii, M., Matsuzaki, I., Chemelli, R. M., Tanaka, H., Williams, S. C., Richarson, J. A., Kozlowski, G. P., Wilson, S., Arch, J. R., Buckingham, R. E., Haynes, A. C., Carr, S. A., Annan, R. S., McNulty, D. E., Liu, W. S., Terrett, J. A., Elshourbagy, N. A., Bergsma, D. J., and Yanagisawa, M. (1998). Orexins and orexin receptors: A family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell 92:573–585.
Schwartz, M. W., Woods, S. C., Porte, D., Seeley, R. J., and Baskin, D. G. (2000). Central nervous system control of food intake. Nature 404:661–671.
Simone, N. L., Bonner, R. F., Gillespie, J. W., Emmert-Buck, M. R., and Liotta, L. A. (1998). Laser-capture microdissection: Opening the microscopic frontier to molecular analysis. Trends Genet. 14:272–276.
Stricker-Krongrad, A., and Beck, B. (2002). Modulation of hypothalamic hypocretin/orexin mRNA expression by glucocorticoids. Biochem. Biophys. Res. Commun. 296:129–133.
Van den Pol, A. N. (1999). Hypothalamic hypocretin (orexin): Robust innervation of the spinal cord. J. Neurosci. 19:3171–3182.
Van der Sluis, P. J., Pool, C. W., and Sluiter, A. A. (1988). Immunochemical detection of peptides and proteins on press-blots after direct tissue gel isoelectric focusing. Electrophoresis 9:654–661.
Vrang, N., Larsen, P. J., Clausen, J. T., and Kristensen, P. (1999). Neurochemical characterization of hypothalamic cocaine-amphetamine-regulated transcript neurons. J. Neurosci. 19:RC5.
Yamanaka, A., Beuckmann, C. T., Willie, J. T., Hara, J., Tsujino, N., Mieda, M., Tominaga, M., Yagami, K., Sugiyama, F., Goto, K., Yanagisawa, M., and Sakurai, T. (2003). Hypothalamic orexin neurons regulate arousal according to energy balance in mice. Neuron 38:701–713.
Yoshida, Y., Fujiki, N., Nakajima, T., Ripley, B., Matsumura, H., Yoneda, H., Mignot, E., and Nishino, S. (2001). Fluctuation of extracellular hypocretin-1 (orexin A) levels in the rat in relation to the light–dark cycle and sleep–wake activities. Eur. J. Neurosci. 14:1075–1081.
Zeitzer, J. M., Buckmaster, C. L., Parker, K. J., Hauck, C. M., Lyons, D. M., and Mignot, E. (2003). Circadian and homeostatic regulation of hypocretin in a primate model: Implications for the consolidation of wakefulness. J. Neurosci. 23:3555–3560.
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Harthoorn, L.F., Sañé, A., Nethe, M. et al. Multi-Transcriptional Profiling of Melanin-Concentrating Hormone and Orexin-Containing Neurons. Cell Mol Neurobiol 25, 1209–1223 (2005). https://doi.org/10.1007/s10571-005-8184-8
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DOI: https://doi.org/10.1007/s10571-005-8184-8