Skip to main content
SpringerLink
Log in

Hyperpolarization-Activated Cation Channels Are Expressed in Rat Hypothalamic Gonadotropin-Releasing Hormone (GnRH) Neurons and Immortalized GnRH Neurons

  • Original Articles
  • Published:
The Journal of the Society for Gynecologic Investigation: JSGI Aims and scope Submit manuscript

Abstract

Objectives

The current research was conducted to determine whether hyperpolarization-activated cyclic nucleotide-gated (HCN1–4) channels are expressed in gonadotropin-releasing hormone (GnRH) neurons in the female rat hypothalamus and immortalized GnRH neurons (GT1–7 cells).

Methods

Double-lavel fluorescence immunohistochemistry was used to colocalized HCN1-4 channels and GnRH in GnRH neurons in the female rat hypothalamus. Reverse transcriptase–polymerase chain reaction (RT-PCR), Westerns blotting, and immunocytochemistry were used to analyze HCN channel gene expression in GT1–7 cells.

Results

Double-label fluorescence immunohistochemistry showed that 43% of hypothalamic GnRH neurons immunostained for HCN2 and 90% of GnRH neurons immunostained for HCN3. RT-PCR and Western blot showed expression of all four HCN channel subunits in GT1–7 cells. Double-label immunochemistry showed cytoplasmic immunostaining of HCN2 and HCN3 in GT1–7 cells.

Conclusions

This study demonstrates for the first time that HCN channels are expressed in GnRH neurons in the rat hypothalamus and GT1–7 cells. Our research supports the hypothesis that HCN channels may be involved in electrical bursting activity and pulsatile GnRH secretion in endogenous GnRH neurons and GT1–7 cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Knobil E, Neil JD. The physiology of reproduction. 2nd ed. New York: Raven Press; 1994.

    Google Scholar 

  2. Terasawa E. Luteinizing hormone-releasing hormone (LHRH) neurons: Mechanism of pulsatile LHRH release. Vitam Horm 2001;63:91–129.

    Article  CAS  PubMed  Google Scholar 

  3. Moenter SM, DeFazio AR, Pitts GR, Nunemaker CS. Mechanisms underlying episodic gonadotropin-releasing hormone secretion. Front Neuroendocrinol 2003;24:79–93.

    Article  CAS  PubMed  Google Scholar 

  4. Terasawa E, Keen KL, Mogi K, Claude P. Pulsatile release of luteinizing hormone-releasing hormone (LHRH) in cultured LHRH neurons derived from the embryonic olfactory placode of the rhesus monkey. Endocrinology 1999;140:1432–41.

    Article  CAS  PubMed  Google Scholar 

  5. Krsmanovic LZ, Martinez-Fuentes AJ, Arora KK, et al. Autocrine regulation of gonadotropin-releasing hormone secretion in cultured hypothalamic neurons. Endocrinology 1999;140:1423–31.

    Article  CAS  PubMed  Google Scholar 

  6. Funabashi T, Daikoku S, Shinohara K, Kimura F. Pulsatile gonadotropin-releasing hormone (GnRH) secretion is an inherent function of GnRH neurons, as revealed by the culture of medial olfactory placode obtained from embryonic rats. Neuroendocrinology 2000;71:138–44.

    Article  CAS  PubMed  Google Scholar 

  7. Martinez de la Escalera G, Choi AL, Weiner RI. Generation and synchronization of gonadotropin-releasing hormone (GnRH) pulses: Intrinsic properties of the GT1-1 GnRH neuronal cell line. Proc Natl Acad Sci U S A 1992;89:1852–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Wetsel WC, Valenca MM, Merchenthaler I, et al. Intrinsic pulsatile secretory activity of immortalized luteinizing hormone-releasing hormone-secreting neurons. Proc Natl Acad Sci U S A 1992;89:4149–53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Krsmanovic LZ, Stojilkovic SS, Merelli F, et al. Calcium signalling and episodic secretion of gonadotropin-releasing hormone in hypothalamic neurons. Proc Natl Acad Sci U S A 1992;89:8462–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Lagrange AH, Ronnekleiv OK, Kelly MJ. Estradiol-17 beta and mu-opioid peptides rapidly hyperpolarize GnRH neurons: A cellular mechanism of negative feedback? Endocrinology 1995;136:2341–4.

    Article  CAS  PubMed  Google Scholar 

  11. Kusano K, Fueshko S, Gainer H, Wray S. Electrical and synaptic properties of embryonic luteinizing hormone-releasing hormone neurons in explant cultures. Proc Natl Acad Sci U S A 1995;92:3918–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Suter KJ, Song WJ, Sampson TL, et al. Genetic targeting of green fluorescent protein to gonadotropin-releasing hormone neurons: Characterization of whole-cell electrophysiological properties and morphology. Endocrinology 2000;141:412–9.

    Article  CAS  PubMed  Google Scholar 

  13. Sim JA, Skynner MJ, Herbison AE. Heterogeneity in the basic membrane properties of postnatal gonadotropin-releasing hormone neurons in the mouse. J Neurosci 2001;21:1067–75.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Kato M, Ui-Tei K, Watanabe M, Sakuma Y. Characterization of voltage-gated calcium currents in gonadotropin-releasing hormone neurons tagged with green fluorescent protein in rats. Endocrinology 2003;144:5118–25.

    Article  CAS  PubMed  Google Scholar 

  15. Abe H, Terasawa E. Firing pattern and rapid modulation of activity by estrogen in primate luteinizing hormone releasing hormone-1 neurons. Endocrinology 2005;146:4312–20.

    Article  CAS  PubMed  Google Scholar 

  16. Suter KH, Wuarin JP, Smith BN, Dudek FE, Moenter SM. Whole-cell recordings from preoptic/hypothalamic slices reveal burst firing in gonadotropin-releasing hormone neurons identified with green fluorescent protein in transgenic mice. Endocrinology 2000;141:3731–6.

    Article  CAS  PubMed  Google Scholar 

  17. Kuehl-Kovarik MC, Pouliot WA, Halterman GL, Handa RJ, Dudek FE, Partin KM. Episodic bursting activity and response to excitatory amino acids in acutely dissociated gonadotropin-releasing hormone neurons genetically targeted with green fluorescent protein. J Neurosci 2002;22:2313–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Bosma MM. Ion channel properties and episodic activity in isolated immortalized gonadotropin-releasing hormone (GnRH) neurons. J Membr Biol 1993;136:85–96.

    Article  CAS  PubMed  Google Scholar 

  19. Van Goor F, Krsmanovic LZ, Catt KJ, Stojilkovic SS. Coordinate regulation of gonadotropin-releasing hormone neuronal firing patterns by cytosolic calcium and store depletion. Proc Natl Acad Sci U S A 1999;96:4101–6.

    Article  Google Scholar 

  20. DiFrancesco D. Pacemaker mechanisms in cardiac tissue. Annu Rev Physiol 1993;55:455–472.

    Article  CAS  PubMed  Google Scholar 

  21. Luthi A, McCormick DA. H-current: Properties of a neuronal and network pacemaker. Neuron 1998;21:9–12.

    Article  CAS  PubMed  Google Scholar 

  22. Maccaferri G, McBain CJ. The hyperpolarization-activated current (Ih) and its contribution to pacemaker activity in rat CA1 hippocampal stratum oriens-alveus interneurones. J Physiol 1996;497:119–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Bal T, McCormick DA. Synchronized oscillations in the inferior olive are controlled by the hyperpolarization-activated cation current I(h). J Neurophysiol 1997;77:3145–56.

    Article  CAS  PubMed  Google Scholar 

  24. Chan CS, Shigemoto R, Mercer JN, Surmeier DJ. HCN2 and HCN1 channels govern the regularity of autonomous pacemaking and synaptic resetting in globus pallidus neurons. J Neurosci 2004;24:9921–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Santoro B, Liu DT, Yao H, et al. Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain. Cell 1998;93:717–29.

    Article  CAS  PubMed  Google Scholar 

  26. Ludwig A, Zong X, Jeglitsch M, Hofmann F, Biel M. A family of hyperpolarization-activated mammalian cation channels. Nature 1998;393:587–91.

    Article  CAS  PubMed  Google Scholar 

  27. Gauss R, Seifert R, Kaupp UB. Molecular identification of a hyperpolarization-activated channel in sea urchin sperm. Nature 1998;393:583–7.

    Article  CAS  PubMed  Google Scholar 

  28. Pape HC. Queern current and pacemaker. The hyperpolarization-activated cation current in neurons. Annu Rev Physiol 1996;58:299–327.

    Article  CAS  PubMed  Google Scholar 

  29. Robinson RB, Siegerbaum SA. Hyperpolarization-activated cation currents: From molecules to physiological function. Annu Rev Physiol 2003;65:453–80.

    Article  CAS  PubMed  Google Scholar 

  30. Monteggia LM, Eisch AJ, Tang MD, Kaczmarek LK, Nestler EJ. Cloning and localization of the hyperpolarization-activated cyclic nucleotide-gated channel family in rat brain. Brain Res Mol Brain Res 2000;81:129–39.

    Article  CAS  PubMed  Google Scholar 

  31. Notomi T, Shigemoto R. Immunohistochemical localization of Ih channel subunits, HCN1-4, in the rat brain. J Comp Neurol 2004;471:241–76.

    Article  CAS  PubMed  Google Scholar 

  32. Mistrik P, Mader R, Michalakis S, Weidinger M, Pfeifer A, Biel M. The murine HCN3 gene encodes a hyperpolarization-activated cation channel with slow kinetics and unique response to cyclic nucleotides. J Biol Chem 2005;280:27056–61.

    Article  CAS  PubMed  Google Scholar 

  33. Mellon PL, Windle JJ, Goldsmith PC, Padula CA, Roberts JL, Weiner RI. Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis. Neuron 1990;5:1–10.

    Article  CAS  PubMed  Google Scholar 

  34. Santoro B, Chen S, Luthi A, et al. Molecular and functional heterogeneity of hyperpolarization-activated pacemaker channels in the mouse CNS. J Neurosci 2000;20:5264–75.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Liposits Z, Merchenthaler I, Wetsel WC, et al. Morphological characterization of immortalized hypothalamic neurons synthesizing luteinizing hormone-releasing hormone. Endocrinology 1991;129:1575–83.

    Article  CAS  PubMed  Google Scholar 

  36. Hsu LJ, Sagara Y, Arroyo A, et al. Alpha-synuclein promotes mitochondrial deficit and oxidative stress. Am J Pathol 2000;157:401–10.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Harris-Warrick RM. Volage-sensitive ion chennels in rhythmic motor systems. Curr Opin Neurobiol 2002;12:646–51.

    Article  CAS  PubMed  Google Scholar 

  38. Ashcroft FM, Rorsman P. Electrophysiology of the pancreatic beta-cell. Prog Biophys Mol Biol 1989;54:87–143.

    Article  CAS  PubMed  Google Scholar 

  39. LeBeau AP, Van Goor F, Stojilkovic SS, Sherman A. Modeling of membrane excitability in gonadotropin-releasing hormone-secreting hypothalamic neurons regulated by Ca2+-mobilizing and adenylyl cyclase-coupled receptors. J Neurosci 2000;20:9290–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Charles A, Weiner R, Costantin J. cAMP modulates the excitability of immortalized H=hypothalamic (GT1) neurons via a cyclic nucleotide gated channel. Mol Endocrinol 2001;15:997–1009.

    Article  CAS  PubMed  Google Scholar 

  41. Oka Y, Matsushima T. Gonadotropin-releasing hormone (GnRH)-immunoreactive terminal nerve cells have intrinsic rhythmicity and project widely in the brain. J Neurosci 1993;13:2161–76.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Oka Y. Tetrodotoxin-resistant persistent Na+ current underlying pacemaker potentials of fish gonadotropin-releasing hormone neurones. J Physiol 1995;482:1–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Abe H, Oka Y. Characterization of K+ currents underlying pacemaker potentials of fish gonadotropin-releasing hormone cells. J Neurophysiol 1999;81:643–53.

    Article  CAS  PubMed  Google Scholar 

  44. Qiu DL, Chu CP, Shirasaka T, et al. Neuromedin U depolarizes rat hypothalamic paraventricular nucleus neurons in vitro by enhancing IH channel activity. J Neurophysiol 2003;90:843–50.

    Article  CAS  PubMed  Google Scholar 

  45. Kamondi A, Reiner PB. Hyperpolarization-activated inward current in histaminergic tuberomammillary neurons of the rat hypothalamus. J Neurophysiol 1991;66:1902–11.

    Article  CAS  PubMed  Google Scholar 

  46. Ghamari-Langroudi M, Bourque CW. Excitatory role of the hyperpolarization-activated inward current in phasic and tonic firing of rat supraoptic neurons. J Neurosci 2000;20:4855–63.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Yu X, Duan XL, Shang CF, Yu HG, Zhou Z. Calcium influx through hyperpolarization-activated cation channels (I(h) channels) contributes to activity-evoked neuronal secretion. Proc Natl Acad Sci U S A 2004;101:1051–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Charles AC, Hales TG. Mechanisms of spontaneous calcium oscillations and action potentials in immortalized hypothalamic (GT1-7) neurons. J Neurophysiol 1995;73:56–64.

    Article  CAS  PubMed  Google Scholar 

  49. Costantin JL, Charles AC. Modulation of Ca(2+) signaling by K(+) channels in a hypothalamic neuronal cell line (GT1-1). J Neurophysiol 2001;85:295–304.

    Article  CAS  PubMed  Google Scholar 

  50. Wetsel WC. Immortalized hypothalamic luteinizing hormone-releasing hormone (LHRH) neurons: A new tool for dissecting the molecular and cellular basis of LHRH physiology. Cell Mol Neurobiol 1995;15:43–78.

    Article  CAS  PubMed  Google Scholar 

  51. Martinez de la Escalera G, Clapp C. Regulation of gonadotropin-releasing hormone secretion: Insights from GT1 immortal GnRH neurons. Arch Med Res 2001;32:486–98.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Gynecology-Obstetrics and Department of Physiology and Biophysics, University at Buffalo, 219 Bryant St., Buffalo, New York, 14222, USA

    Armando Arroyo MD, Beomsu Kim PhD, Randall L. Rasmusson PhD, Glenna Bett PhD & John Yeh MD

Authors
  1. Armando Arroyo MD
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Beomsu Kim PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Randall L. Rasmusson PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Glenna Bett PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. John Yeh MD
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Armando Arroyo MD.

Additional information

Supported in part by the University at Buffalo, The State University of New York (SUNY), Interdisciplinary Research Creative Activities Fund (IRCAF).

The authors thank Jennifer Peresie for technical assistance and Dr Wade Sigurdson, Director of the Confocal Microscopy Center at University at Buffalo School of Medicine and Biomedical Sciences, for assistance with confocal imaging.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Arroyo, A., Kim, B., Rasmusson, R.L. et al. Hyperpolarization-Activated Cation Channels Are Expressed in Rat Hypothalamic Gonadotropin-Releasing Hormone (GnRH) Neurons and Immortalized GnRH Neurons. Reprod. Sci. 13, 442–450 (2006). https://doi.org/10.1016/j.jsgi.2006.05.010

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1016/j.jsgi.2006.05.010

Key words

Search

Navigation