Elsevier

Cell Calcium

Volume 51, Issues 3–4, March–April 2012, Pages 267-276
Cell Calcium

Understanding calcium homeostasis in postnatal gonadotropin-releasing hormone neurons using cell-specific Pericam transgenics

https://doi.org/10.1016/j.ceca.2011.11.005Get rights and content

Abstract

The gonadotropin-releasing hormone (GnRH) neurons are the key output cells of a complex neuronal network controlling fertility in mammals. To examine calcium homeostasis in postnatal GnRH neurons, we generated a transgenic mouse line in which the genetically encodable calcium indicator ratiometric Pericam (rPericam) was targeted to the GnRH neurons. This mouse model enabled real-time imaging of calcium concentrations in GnRH neurons in the acute brain slice preparation. Investigations in GnRH-rPericam mice revealed that GnRH neurons exhibited spontaneous, long-duration (∼8 s) calcium transients. Dual electrical-calcium recordings revealed that the calcium transients were correlated perfectly with burst firing in GnRH neurons and that calcium transients in GnRH neurons regulated two calcium-activated potassium channels that, in turn, determined burst firing dynamics in these cells. Curiously, the occurrence of calcium transients in GnRH neurons across puberty or through the estrous cycle did not correlate well with the assumption that GnRH neuron burst firing was contributory to changing patterns of pulsatile GnRH release at these times. The GnRH-rPericam mouse was also valuable in determining differential mechanisms of GABA and glutamate control of calcium levels in GnRH neurons as well as effects of G-protein-coupled receptors for GnRH and kisspeptin. The simultaneous measurement of calcium levels in multiple GnRH neurons was hampered by variable rPericam fluorescence in different GnRH neurons. Nevertheless, in the multiple recordings that were achieved no evidence was found for synchronous calcium transients. Together, these observations show the great utility of transgenic targeting strategies for investigating the roles of calcium with specified neuronal cell types.

Section snippets

Central control of fertility

Fertility is the physiological readout of the activity of the hypothalamic–pituitary–gonadal (HPG) axis [1]. In all mammals examined to date, gonadotropin-releasing hormone (GnRH) is the ultimate output of the central neuronal network that regulates fertility. Secreted by axon terminals in close apposition with the fenestrated capillary bed of the hypophyseal portal vasculature, GnRH reaches the anterior pituitary gonadotropes to regulate the secretion of luteinizing hormone (LH) and

Development of GnRH neuron transgenics

The scattered spatial distribution of the GnRH neuron cell population has proven to be the main obstacle to understanding the GnRH neuron. Most likely as a consequence of the unique migration of GnRH neurons from the nose into the brain during gestation [10], [11], the GnRH neuron cell bodies are scattered throughout the basal forebrain from the medial septum through to the base of the hypothalamus [12]. This has meant that not only are GnRH neurons very difficult to pre-identify in brain slice

Brief overview of calcium control in neurons

Calcium is the most versatile intracellular messenger [27]. The majority of cell-to-cell communication evokes fluctuations in intracellular calcium concentration ([Ca2+]i). Furthermore, because cells typically maintain a very low [Ca2+]i under basal conditions, it is typical for [Ca2+]i to rise more than 100-times upon stimulation, thus providing a sensitive metric of changes in cell physiology.

Increases in [Ca2+]i can occur through the influx of external calcium or calcium release from

Development of GnRH neuron-Pericam transgenics

A number of approaches had been taken to monitor Ca2+ dynamics directly in neurons, including bulk loading with acetoxymethyl ester calcium indicators in brain slices [39], [40], [41], [42], [43]. Although these methods are viable for monitoring Ca2+ dynamics in embryonic GnRH neurons in culture [26], [44], [45], they are less effective in brain tissue in vivo [46] and are further hampered by the scattered distribution of GnRH neurons within the brain. However, the key objective has always been

GnRH neurons exhibit robust calcium transients

Monitoring individual adult GnRH-rPericam neurons with time-lapse calcium imaging unveiled transient elevations in [Ca2+]i in the majority (∼70%) of recorded cells ([54]; Fig. 2). This was the first observation of spontaneous [Ca2+]i transients in neurons of the adult brain. Even more surprisingly, each transient was of very long duration lasting ∼8 s (Fig. 2B and C). The transients could be observed in individual GnRH neurons at different frequencies but, on average, were found to occur every

GnRH neuron calcium transients originate from a dual action of VGCC and store release initiated by action potential firing

We were immediately intrigued to understand the origin of the long [Ca2+]i transients in GnRH neurons. Adult mouse GnRH neurons express the full panel of voltage-gated calcium channels (L-, R-, P/Q-, and T-type) [13], [68], [69], [70], [71]. Thus, a role of VGCC was probable as was an involvement of internal stores, as early studies showed that a non-specific InsP3R antagonist 2-APB suppressed transients in GnRH neurons [54].

To examine this mechanism in detail we developed a brain slice

Role of calcium transients in GnRH neurons

Although [Ca2+]i transients undoubtedly have multiple roles in GnRH neuron physiology, it appears that electrical activity is one cell behavior tightly regulated by calcium. For example, the burst firing dynamics of GnRH neurons were altered dramatically in calcium-depleted extracellular medium [72]. While the regulation of GnRH neuron burst duration by apamin-sensitive calcium-activated potassium (SK) channels had been described earlier [73], [74], the real complexity of the interplay between

[Ca2+]i transient synchronization in GnRH neurons

Given that [Ca2+]i transients represent a good proxy for bursting behavior in GnRH neurons, we were interested in using the GnRH rPericam mice to examine the electrical behavior of multiple GnRH neurons at the same time. However, this initiative was not especially successful. First, each brain slice was found to have relatively few GnRH neurons with sufficient rPericam fluorescence to enable reliable recording. This, coupled with the rather low chance of getting several of these

Changes in [Ca2+]i transients under different physiological conditions

The majority of recordings from GnRH-rPericam mice reported above were obtained from male mice that exhibit a relatively continuous pulsatile pattern of GnRH and LH secretion [82]. In one study [72] we also examined the calcium dynamics of GnRH neurons obtained from diestrous female mice, also exhibiting regular GnRH/LH pulses, and found these to be similar to those of male mice. We were interested, therefore, to determine whether animals exhibiting different modes of LH secretion would show

Ionotropic GABA and glutamate receptors

Electrical recordings from GnRH neurons in situ show that the vast majority of afferent inputs to these neurons signal through ionotropic GABAA and glutamate receptors (for review see refs. [86], [87]). We were interested, therefore, in understanding what impact these neurotransmitter would have on [Ca2+]i in GnRH neurons.

The responses of GnRH neurons were assessed by exogenous application of GABA and two agonists of glutamate receptors, 2-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)

Conclusions

rPericam was one of the first successful GECIs enabling real-time monitoring of [Ca2+]i in specific neuronal phenotypes. The GnRH-rPericam mouse has been invaluable in our hands in enabling real-time monitoring of [Ca2+]i in GnRH neurons in the acute brain slice preparation. The mouse has led to the discovery of spontaneous [Ca2+]i transients in mature GnRH neurons and a clear understanding of how they are generated. The dual calcium-electrical recording paradigm has been invaluable in

Acknowledgements

The authors wish to thank all those in the Herbison laboratory who have contributed to these series of experiments. Funding was provided by the UK Wellcome Trust, NZ Marsden Fund and NZ Health Research Council.

References (96)

  • M.J. Berridge

    Calcium microdomains: organization and function

    Cell Calcium

    (2006)
  • R. Yuste et al.

    Control of postsynaptic Ca2+ influx in developing neocortex by excitatory and inhibitory neurotransmitters

    Neuron

    (1991)
  • M. Osanai et al.

    Long-lasting spontaneous calcium transients in the striatal cells

    Neurosci. Lett.

    (2006)
  • M. Canepari et al.

    GABA- and glutamate-mediated network activity in the hippocampus of neonatal and juvenile rats revealed by fast calcium imaging

    Cell Calcium

    (2000)
  • O. Griesbeck

    Fluorescent proteins as sensors for cellular functions

    Curr. Opin. Neurobiol.

    (2004)
  • O. Garaschuk et al.

    Troponin C-based biosensors: a new family of genetically encoded indicators for in vivo calcium imaging in the nervous system

    Cell Calcium

    (2007)
  • T. Knopfel et al.

    Optical probing of neuronal circuit dynamics: genetically encoded versus classical fluorescent sensors

    Trends Neurosci.

    (2006)
  • N.C. Spitzer et al.

    Orchestrating neuronal differentiation: patterns of Ca2+ spikes specify transmitter choice

    Trends Neurosci.

    (2004)
  • A.C. Charles et al.

    Intercellular calcium waves in neurons

    Mol. Cell. Neurosci.

    (1996)
  • J. Clarkson et al.

    Development of GABA and glutamate signaling at the GnRH neuron in relation to puberty

    Mol. Cell. Endocrinol.

    (2006)
  • J. Clarkson et al.

    Neurobiological mechanisms underlying kisspeptin activation of gonadotropin-releasing hormone (GnRH) neurons at puberty

    Mol. Cell. Endocrinol.

    (2010)
  • S.M. Moenter et al.

    Mechanisms underlying episodic gonadotropin-releasing hormone secretion

    Front. Neuroendocrinol.

    (2003)
  • K. Iremonger et al.

    Glutamate regulation of GnRH neuron excitability

    Brain Res.

    (2010)
  • J.M. Castellano et al.

    Ontogeny and mechanisms of action for the stimulatory effect of kisspeptin on gonadotropin-releasing hormone system of the rat

    Mol. Cell. Endocrinol.

    (2006)
  • T.M. Plant

    Puberty in primates

  • E. Terasawa

    Control of luteinizing hormone-releasing hormone pulse generation in nonhuman primates

    Cell. Mol. Neurobiol.

    (1995)
  • I.J. Clarke et al.

    GnRH pulse frequency determines LH pulse amplitude by altering the amount of releasable LH in the pituitary glands of ewes

    J. Reprod. Fertil.

    (1985)
  • P.E. Belchetz et al.

    Hypophysial responses to continuous and intermittent delivery of hypopthalamic gonadotropin-releasing hormone

    Science

    (1978)
  • E. Knobil et al.

    Control of the rhesus monkey menstrual cycle: permissive role of hypothalamic gonadotropin-releasing hormone

    Science

    (1980)
  • T.W. Valk et al.

    Hypogonadotropic hypogonadism: hormonal responses to low dose pulsatile administration of gonadotropin-releasing hormone

    J. Clin. Endocrinol. Metab.

    (1980)
  • C.A. Christian et al.

    The neurobiology of preovulatory and estradiol-induced gonadotropin-releasing hormone surges

    Endocr. Rev.

    (2010)
  • M. Schwanzel-Fukuda et al.

    Origin of luteinizing hormone-releasing hormone neurons

    Nature

    (1989)
  • S. Wray et al.

    Evidence that cells expressing luteinizing hormone-releasing hormone mRNA in the mouse are derived from progenitor cells in the olfactory placode

    Proc. Natl. Acad. Sci. U.S.A.

    (1989)
  • A.J. Silverman et al.

    The gonadotropin-releasing hormone (GnRH), neuronal systems: immunocytochemistry and in situ hybridization

  • J.A. Sim et al.

    Heterogeneity in the basic membrane properties of postnatal gonadotropin-releasing hormone neurons in the mouse

    J. Neurosci.

    (2001)
  • M.J. Skynner et al.

    Promoter transgenics reveal multiple gonadotropin-releasing hormone-I-expressing cell populations of different embryological origin in mouse brain

    J. Neurosci.

    (1999)
  • D.J. Spergel et al.

    GABA- and glutamate-activated channels in green fluorescent protein-tagged gonadotropin-releasing hormone neurons in transgenic mice

    J. Neurosci.

    (1999)
  • K.J. Suter et al.

    Genetic targeting of green fluorescent protein to gonadotropin-releasing hormone neurons: characterization of whole-cell electrophysiological properties and morphology

    Endocrinology

    (2000)
  • S.K. Han et al.

    Endogenous GABA release inhibits the firing of adult gonadotropin-releasing hormone neurons

    Endocrinology

    (2004)
  • H. Fujioka et al.

    Generation of transgenic rats expressing enhanced green fluorescent protein in gonadotropin-releasing hormone neurons

    J. Reprod. Dev.

    (2003)
  • J.A. Sim et al.

    Late postnatal reorganization of GABA(A) receptor signalling in native GnRH neurons

    Eur. J. Neurosci.

    (2000)
  • M.J. Skynner et al.

    Detection of estrogen receptor alpha and beta messenger ribonucleic acids in adult gonadotropin-releasing hormone neurons

    Endocrinology

    (1999)
  • R.E. Campbell et al.

    Biocytin filling of adult gonadotropin-releasing hormone neurons in situ reveals extensive, spiny, dendritic processes

    Endocrinology

    (2005)
  • E.C Cottrell et al.

    Postnatal re-modelling of dendritic structure and spine density in gonadotropin-releasing hormone (GnRH) neurons

    Endocrinology

    (2006)
  • H. Chan et al.

    Dendritic spine plasticity in gonadatropin-releasing hormone (GnRH) neurons activated at the time of the preovulatory surge

    Endocrinology

    (2011)
  • S. Constantin

    Physiology of the GnRH neuron: studies from embryonic GnRH neurons

    J. Neuroendocrinol.

    (2011)
  • M.J. Berridge et al.

    The versatility and universality of calcium signalling

    Nat. Rev. Mol. Cell Biol.

    (2000)
  • T.A. Koshimizu et al.

    Characterization of calcium signaling by purinergic receptor-channels expressed in excitable cells

    Mol. Pharmacol.

    (2000)

    • Cited by (16)

    • How does apelin affect LH levels? An investigation at the level of GnRH and KNDy neurons

      2022, Molecular and Cellular Endocrinology
      Citation Excerpt :

      The second reason is related to apelin signaling: apelin binding to APJ receptor induces activation of Gαq/11 or Gαi/o and thus can modify intracellular calcium dynamics (Masri et al., 2006; Szokodi et al., 2002). When recorded in vitro or in vivo, a proportion of GnRH neurons exhibited [Ca2+]i transients lasting several seconds (Constantin et al., 2009, 2012). Recording simultaneously GnRH neurons electrical activity and intracellular calcium in neurons showed a robust correlation between bursts of action potentials (APs) and [Ca2+]i transients (Lee et al., 2010) whereas GnRH neurons presenting APs without a bursting pattern did not display [Ca2+]i transients.

    • Generation of kisspeptin-responsive GnRH neurons from human pluripotent stem cells

      2017, Molecular and Cellular Endocrinology
      Citation Excerpt :

      This is the first report of in vitro generated GnRH neurons displaying spontaneous and kisspeptin-regulated calcium transients. The production of spontaneous calcium transients whose frequency and amplitude can be modulated by neuropeptides such as kisspeptin is a hallmark of GnRH neurons (Constantin et al., 2009, 2012; Han et al., 2005). In accordance with this, about 20% of our differentiated neurons displayed both spontaneous and kisspeptin-stimulated calcium transients.

    • Physiology of the Adult Gonadotropin-Releasing Hormone Neuronal Network

      2015, Knobil and Neill's Physiology of Reproduction: Two-Volume Set

    • MRNA expression of ion channels in GnRH neurons: Subtype-specific regulation by 17β-estradiol

      2013, Molecular and Cellular Endocrinology
      Citation Excerpt :

      Our previous results comparing T-type ion channel mRNA expression and current density in GnRH neurons and KCNQ channel mRNAs to M-current in NPY neurons, have found a close correlation between alterations in mRNA expression and function (Roepke et al., 2011; Zhang et al., 2009). Numerous studies have illustrated that calcium entry through voltage-gated channels contributes to neuronal excitability and is important for GnRH neuronal excitability and GnRH release (Constantin et al., 2010, 2011; Iremonger and Herbison, 2012; Kroll et al., 2011; Krsmanovic et al., 1992; Lee et al., 2010; Sun et al., 2010; Zhang et al., 2009; Zhang and Spergel, 2012). However, more studies are needed to explore the correlation between HVA channel expression and HVA currents in GnRH neurons under different experimental conditions.

    • Neuroendocrine signalling: Natural variations on a Ca<sup>2+</sup> theme

      2012, Cell Calcium

    • Gonadal Feedback Alters the Relationship between Action Potentials and Hormone Release in Gonadotropin-Releasing Hormone Neurons in Male Mice

      2023, Journal of Neuroscience
    View all citing articles on Scopus
    1

    These authors contributed equally.

    View full text
    Copyright © 2011 Elsevier Ltd. All rights reserved.