Assessment of the Role of the Glutamatergic System in the Activation of Melanin-Concentrating Hormone Neurons

Abstract

The hypothalamus is the part of the central nervous system that contains many neuropeptide and neurotransmitter systems. It functions as an integrative center for peripheral and central signals that control appetite and regulate energy homeostasis. Neurons located in various nuclei of the hypothalamus regulate feeding behavior with the many different hormones and peptides they synthesize. While some of these molecules have orexigenic effects that stimulate food intake, others have anorexigenic effects that suppress food intake. One orexigenic peptide is melanin-concentrating hormone (MCH). The mammalian melanin-concentrating hormone is a nineteen-amino acid neuropeptide produced in the lateral hypothalamic area (LHA) and incerto-hypothalamic area (IHA) in the brain and exerts its orexigenic effects through MCHR1 receptors. In our study, we investigated the relationship between the glutamatergic system and MCH neurons histologically. In the experiments, double immunohistochemical labeling with anti-MCH and anti-pCREB antibodies was performed on sections from rat brains injected with glutamate agonists and antagonists. CREB phosphorylation (pCREB) was used as a marker of neuronal activation in the experiments. In male rats, an increased proportion of MCH neurons expressing pCREB was observed in the lateral hypothalamic nucleus after injection of all three agonists. Applying the specific antagonist before injections of all three agonists led to a statistically significant decrease in the number of activated MCH neurons. This study shows that intracellular pathways that require p-CREB protein expression are used to activate MCH neurons. Additionally, for the first time in the literature, the presence of phosphorylated CREB protein in MCH neurons was determined. New information has been added to the literature on glutamatergic regulation of MCH neurons.

Keywords

• Melanin-concentrating hormone (MCH)
• pCREB
• Glutamate
• Hypothalamus
• Immunohistochemistry
• Rat

Glutamaterjik Sistemin Melanin Yoğunlaştırıcı Hormon Nöronlarının Aktivasyonundaki Rolünün Değerlendirilmesi

Abstract

Hipotalamus, çok sayıda nöropeptit ve nörotransmitter sistemin yer aldığı merkezi sinir sistemi bölümüdür. İştahın kontrol edilmesi ve enerji homeostazisinin düzenlenmesini sağlayan periferal ve merkezi sinyaller için integratif bir merkez olarak fonksiyon görür. Hipotalamusun çeşitli çekirdeklerinde yerleşik nöronlar, sentezledikleri çok sayıda farklı hormon ve/veya peptit ile beslenme davranışı üzerinde düzenleme yapmaktadırlar. Bu moleküllerin bir kısmı besin alımını tetikleyici oreksijenik etki gösterirken, bir bölümü de baskılayıcı anoreksijenik etkiye sahiptirler. Oreksijenik peptitlerden biri de melanin konsantre edici hormondur (MCH). Memeli MCH'ı, beyinde lateral hipotalamik alan (LHA) ile inserto-hipotalamik alanda (IHA) üretilen on dokuz amino asitli bir nöropeptiddir ve oreksijenik etkilerini MCHR1 reseptörleri üzerinden göstermektedir. Çalışmamızda glutamaterjik sistemle MCH nöronları arasındaki ilişki histolojik olarak araştırılmıştır. Deneylerde glutamat agonisti ve antagonisti verilen sıçan beyinlerinden elde edilen kesitlere, anti-MCH ve anti-pCREB antikorlarıyla ikili immünohistokimyasal işaretlemeler yapılmıştır. Çalışmalarda CREB fosforilasyonu (pCREB) nöronal aktivasyon belirteci olarak kullanılmıştır. Erkek deneklerde, lateral hipotalamik çekirdekte her üç agonistin enjeksiyonu sonrasında pCREB’i eksprese eden MCH nöronlarının oranının arttığı gözlenmiştir. Her üç agonistin enjeksiyonlarından önce özgün antagonist uygulanması, aktive olan MCH nöron sayısında istatistiksel olarak anlamlı bir azalmaya yol açmıştır. Bu çalışma ile MCH nöronlarının aktivasyonunda p-CREB protein ekspresyonunu gerektiren hücre içi yolakların kullanıldığı gösterilmiştir. Ayrıca literatürde ilk kez MCH nöronlarında fosforile CREB proteininin varlığı belirlenmiştir. MCH nöronlarının glutamaterjik regülasyonu açısından literatüre yeni bilgiler kazandırılmıştır.

Keywords

• Melanin konsantre edici hormon (MCH)
• p-CREB
• Glutamat
• Hipotalamus
• İmmünohistokimya
• Sıçan

References

1. 1. Ruggiero-Ruff RE, Coss D. Neuroendocrinology and the Genetics of Obesity. Endocrinology. 2025;166(9):bqaf121. doi:10.1210/endocr/bqaf121
2. 2. Bittencourt JC, Presse F, Arias C. The melanin-concentrating hormone system of the rat brain: an immuno- and hybridization histochemical characterization. J Comp Neurol. 1992;319:218–245.
3. 3. Chen X, Wang Y, Fu S, et al. The Integrated Function of the Lateral Hypothalamus in Energy Homeostasis. Cells. 2025;14(14):1042. Published 2025 Jul 8. doi:10.3390/cells14141042
4. 4. Sears RM, Liu RJ, Narayanan NS, et al. Regulation of nucleus accumbens activity by the hypothalamic neuropeptide melanin-concentrating hormone. J Neurosci. 2010;30:8263–8273.
5. 5. Guyon A, Conductier G, Rovere C, Enfissi A, Nahon JL. Melanin-concentrating hormone producing neurons: activities and modulations. Peptides. 2009;30(11):2031–2039.
6. 6. Williams G, Bing C, Cai XJ, et al. The hypothalamus and the control of energy homeostasis: different circuits, different purposes. Physiol Behav. 2001;74:683–701.
7. 7. Qu D, Ludwig DS, Gammeltoft S, et al. A role for melanin-concentrating hormone in the central regulation of feeding behaviour. Nature. 1996;380:243–247.
8. 8. Concetti C, Peleg-Raibstein D, Burdakov D. Hypothalamic MCH Neurons: From Feeding to Cognitive Control. Function (Oxf). 2023;5(1):zqad059. Published 2023 Oct 27. doi:10.1093/function/zqad059

9. 9. Williams G, Cai XJ, Elliott JC, et al. Anabolic neuropeptides. Physiol Behav. 2004;81:211–222.
10. 10. Harthoorn LF, Sane A, Nethe M, van Heerikhuize JJ. Multi-transcriptional profiling of melanin-concentrating hormone and orexin-containing neurons. Cell Mol Neurobiol. 2005;25(8):1209–1223.
11. 11. Toossi H, Del Cid-Pellitero E, Jones BE. GABA receptors on orexin and melanin-concentrating hormone neurons are differentialy homeostatically regulated following sleep deprivation. eNeuro. 2016;3(3):ENEURO.0077-16.2016.
12. 12. Bartolotti N, Lazarov O. CREB signals as PBMC-based biomarkers of cognitive dysfunction: A novel perspective of the brain-immune axis. Brain Behav Immun. 2019;78:9-20. doi:10.1016/j.bbi.2019.01.004
13. 13.Eyigor O, Minbay Z, Cavusoglu I. Activation of orexin neuronsthrough non-NMDA glutamate receptors evidenced by c-Fosimmunohistochemistry. Endocrine. 2010;37(1):167-172. doi:10.1007/s12020-009-9284-x
14. 14.Gok-Yurtseven D, Kafa IM, Minbay Z, Eyigor O.Glutamatergic activation of A1 and A2 noradrenergic neuronsin the rat brain stem. Croat Med J. 2019;60(4):352-360. doi:10.3325/cmj.2019.60.352
15. 15.Serter Kocoglu S, Gok Yurtseven D, Cakir C, Minbay Z,Eyigor O. Glutamatergic Activation of Neuronostatin Neuronsin the Periventricular Nucleus of the Hypothalamus. Brain Sci.2020;10(4):217. Published 2020 Apr 6.doi:10.3390/brainsci10040217
16. 16.Gok Yurtseven D, Serter Kocoglu S, Minbay Z, Eyigor O.Immunohistochemical Evidence for Glutamatergic Regulationof Nesfatin-1 Neurons in the Rat Hypothalamus. Brain Sci.2020;10(9):630. Published 2020 Sep 11.doi:10.3390/brainsci10090630
17. 17.Paxinos G, Watson C. The rat brain in stereotaxic coordinates. 6th ed. Amsterdam: Elsevier Academic Press; 2009.
18. 18.Chometton S, Croizier S, Fellmann D, et al. The MCH neuronpopulation as a model for the development and evolution of thelateral and dorsal hypothalamus. J Chem Neuroanat. 2016;75:28–31.
19. 19.Lord MN, Subramanian K, Kanoski SE, Noble EE. Melanin-concentrating hormone and food intake control: sites of action, peptide interactions, and appetition. Peptides. 2021;137:170476.
20. 20.Shi L, He Y, Lian Y, Luo J, Zhu X, Zhao H. Melanin-concentrating hormone: A promising target for antidepressanttreatment. Pharmacol Biochem Behav. 2025;250:173999.doi:10.1016/j.pbb.2025.173999
21. 21.Della-Zuana O, Presse F, Ortola C, et al. Acute and chronicadministration of melanin-concentrating hormone enhancesfood intake and body weight in Wistar and Sprague-Dawley rats. Int J Obes Relat Metab Disord. 2002;26(10):1289–1295.
22. 22.Whitlock BK, Daniel JA, McMahon CD, et al.Intracerebroventricular melanin-concentrating hormonestimulates food intake in sheep. Domest Anim Endocrinol. 2005;28(2):224–232.
23. 23.Gomori A, Ishihara A, Ito M, et al. Chronicintracerebroventricular infusion of MCH causes obesity in mice. Am J Physiol Endocrinol Metab. 2003;284(3):E583–E588.
24. 24.Ludwig DS, Tritos NA, Mastaitis JW, et al. Melanin-concentrating hormone overexpression in transgenic mice leadsto obesity and insulin resistance. J Clin Invest.2001;107(3):379–386.
25. 25.Chee MJ, Pissios P, Maratos-Flier E. Neurochemicalcharacterization of neurons expressing melanin-concentrating hormone receptor 1 in the mouse hypothalamus. J CompNeurol. 2013;521(10):2208–2234.
26. 26.Haemmerle CA, Campos AM, Bittencourt JC. Melanin-concentrating hormone inputs to the nucleus accumbensoriginate from distinct hypothalamic sources and are apposed to GABAergic and cholinergic cells in the Long-Evans rat brain.Neuroscience. 2015;289:392–405.
27. 27.Terrill SJ, Subramanian KS, Lan R, et al. Nucleus accumbensmelanin-concentrating hormone signaling promotes feeding in a sex-specific manner. Neuropharmacology. 2020;178:108270.
28. 28.Abbott CR, Kennedy AR, Wren AM, et al. Identification ofhypothalamic nuclei involved in the orexigenic effect ofmelanin-concentrating hormone. Endocrinology.2003;144(9):3943–3949.
29. 29.Payant MA, Spencer CD, Ly NKK, Chee MJ. Inhibitory actions of melanin-concentrating hormone in the lateral septum. JPhysiol. 2024;602(14):3545-3574. doi:10.1113/JP284845
30. 30.Zhang L, Zhao H, Zhang X, et al. Nobiletin protects againstcerebral ischemia via activating the p-Akt, p-CREB, BDNF and Bcl-2 pathway and ameliorating BBB permeability in rat. BrainRes Bull. 2013;96:45-53. doi:10.1016/j.brainresbull.2013.04.009
31. 31.De Cesare D, Fimia GM, Sassone-Corsi P. Signaling routes toCREM and CREB: plasticity in transcriptional activation.Trends Biochem Sci. 1999;24(7):281-285. doi:10.1016/s0968-0004(99)01414-0
32. 32.Montminy M. Transcriptional regulation by cyclic AMP. Annu Rev Biochem. 1997;66:807-822. doi:10.1146/annurev.biochem.66.1.807
33. 33.Redmond L, Kashani AH, Ghosh A. Calcium regulation ofdendritic growth via CaM kinase IV and CREB-mediated transcription. Neuron. 2002;34(6):999-1010. doi:10.1016/s0896-6273(02)00737-7