scholarly journals Suppression of Basal Spontaneous Gonadotropin-Releasing Hormone Neuronal Activity during Lactation: Role of Inhibitory Effects of Neuropeptide Y

Endocrinology ◽  
2008 ◽  
Vol 150 (1) ◽  
pp. 333-340 ◽  
Author(s):  
Jing Xu ◽  
Melissa A. Kirigiti ◽  
Michael A. Cowley ◽  
Kevin L. Grove ◽  
M. Susan Smith
Keyword(s):  
Neuropeptide Y ◽  
Neuronal Activity ◽  
Fluorescent Protein ◽  
Resting Membrane Potential ◽  
Inhibitory Effects ◽  
Gnrh Neurons ◽  
Cell Current ◽  
Green Fluorescent ◽  
Hypothalamic Slices

Increased neuropeptide Y (NPY) activity drives the chronic hyperphagia of lactation and may contribute to the suppression of GnRH activity. The majority of GnRH neurons are contacted by NPY fibers, and GnRH cells express NPY Y5 receptor (Y5R). Therefore, NPY provides a neurocircuitry for information about food intake/energy balance to be directly transmitted to GnRH neurons. To investigate the effects of lactation on GnRH neuronal activity, hypothalamic slices were prepared from green fluorescent protein-GnRH transgenic rats. Extracellular loose-patch recordings determined basal GnRH neuronal activity from slices of ovariectomized control and lactating rats. Compared with controls, hypothalamic slices from lactating rats had double the number of quiescent GnRH neurons (14.51 ± 2.86 vs. 7.04 ± 2.84%) and significantly lower firing rates of active GnRH neurons (0.25 ± 0.02 vs. 0.37 ± 0.03 Hz). To study the NPY-postsynaptic Y5R system, whole-cell current-clamp recordings were performed in hypothalamic slices from control rats to examine NPY/Y5R antagonist effects on GnRH neuronal resting membrane potential. Under tetrodotoxin treatment, NPY hyperpolarized GnRH neurons from −56.7 ± 1.94 to −62.1 ± 1.83 mV; NPY’s effects were blocked by Y5R antagonist. To determine whether increased endogenous NPY tone contributes to GnRH neuronal suppression during lactation, hypothalamic slices were treated with Y5R antagonist. A significantly greater percentage of GnRH cells were activated in slices from lactating rats (52%) compared with controls (28%). These results suggest that: 1) basal GnRH neuronal activity is suppressed during lactation; 2) NPY can hyperpolarize GnRH neurons via postsynaptic Y5R; and 3) increased inhibitory NPY tone during lactation is a component of the mechanisms responsible for suppression of GnRH neuronal activity. Neuropeptide Y (NPY) directly hyperpolarizes GnRH neurons via postsynaptic NPY Y5 receptor. Increased inhibitory NPY tone during lactation is an important component of the suppression of GnRH neuronal activity.

scholarly journals 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 ◽  
Vol 141 (10) ◽  
pp. 3731-3736 ◽  
Author(s):  
Kelly J. Suter ◽  
Jean-Pierre Wuarin ◽  
Bret N. Smith ◽  
F. Edward Dudek ◽  
Suzanne M. Moenter
Keyword(s):  
Green Fluorescent Protein ◽  
High Frequency ◽  
Action Potentials ◽  
Pulse Generator ◽  
Fluorescent Protein ◽  
Neuroendocrine Cells ◽  
Gnrh Neurons ◽  
Green Fluorescent ◽  
Whole Cell Recordings ◽  
Hypothalamic Slices

Abstract Central control of reproduction is governed by a neuronal pulse generator that underlies the activity of hypothalamic neuroendocrine cells that secrete GnRH. Bursts and prolonged episodes of repetitive action potentials have been associated with hormone secretion in this and other neuroendocrine systems. To begin to investigate the cellular mechanisms responsible for the GnRH pulse generator, we used transgenic mice in which green fluorescent protein was genetically targeted to GnRH neurons. Whole-cell recordings were obtained from 21 GnRH neurons, visually identified in 200-μm preoptic/hypothalamic slices, to determine whether they exhibit high frequency bursts of action potentials and are electrically coupled at or near the somata. All GnRH neurons fired spontaneous action potentials, and in 15 of 21 GnRH neurons, the action potentials occurred in single bursts or episodes of repetitive bursts of high frequency spikes (9.77 ± 0.87 Hz) lasting 3–120 sec. Extended periods of quiescence of up to 30 min preceded and followed these periods of repetitive firing. Examination of 92 GnRH neurons (including 32 neurons that were located near another green fluorescent protein-positive neuron) revealed evidence for coupling in only 1 pair of GnRH neurons. The evidence for minimal coupling between these neuroendocrine cells suggests that direct soma to soma transfer of information, through either cytoplasmic bridges or gap junctions, has a minor role in synchronization of GnRH neurons. The pattern of electrical activity observed in single GnRH neurons within slices is temporally consistent with observations of GnRH release and multiple unit electrophysiological correlates of LH release. Episodes of burst firing of individual GnRH neurons may represent a component of the GnRH pulse generator.

scholarly journals Origin of Neuropeptide Y-Containing Afferents to Gonadotropin-Releasing Hormone Neurons in Male Mice

Endocrinology ◽  
2003 ◽  
Vol 144 (11) ◽  
pp. 4967-4974 ◽  
Author(s):  
Gergely F. Turi ◽  
Zsolt Liposits ◽  
Suzanne M. Moenter ◽  
Csaba Fekete ◽  
Erik Hrabovszky
Keyword(s):  
Green Fluorescent Protein ◽  
Neuropeptide Y ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Monosodium Glutamate ◽  
Male Mice ◽  
Combined Application ◽  
Gnrh Neurons ◽  
Arcuate Nuclei ◽  
Green Fluorescent

Abstract The origin of neuropeptide Y (NPY) afferents to GnRH neurons was investigated in male mice. Neonatal lesioning of the hypothalamic arcuate nuclei (ARC) with monosodium glutamate markedly reduced the number of NPY fibers in the preoptic area as well as the frequency of their contacts with perikarya and proximal dendrites of GnRH neurons. Dual-label immunofluorescence studies to determine the precise contribution of the ARC to the innervation of GnRH neurons by NPY axons were carried out on transgenic mice in which enhanced green fluorescent protein was expressed under the control of the GnRH promoter (GnRH-enhanced green fluorescent protein mice). The combined application of red Cy3 and blue AMCA fluorochromogenes established that 49.1 ± 7.3% of NPY axons apposed to green GnRH neurons also contained agouti-related protein (AGRP), a selective marker for NPY axons arising from the ARC. Immunoelectronmicroscopic analysis detected symmetric synapses between AGRP fibers and GnRH-immunoreactive perikarya. Additional triple-fluorescence experiments revealed the presence of dopamine-β-hydroxylase immunoreactivity within 25.4 ± 3.3% of NPY afferents to GnRH neurons. This enzyme marker enabled the selective labeling of NPY pathways ascending from noradrenergic/adrenergic cell populations of the brain stem, thus defining a second important source for NPY-containing fibers regulating GnRH cells. The absence of both topographic markers (AGRP and dopamine-β-hydroxylase) within 26% of NPY contacts suggests that additional sources of NPY fibers to GnRH neurons exist. Future studies will address distinct functions of the two identified NPY systems in the afferent neuronal regulation of the GnRH system.

scholarly journals An inhibitory circuit from brainstem to GnRH neurons in male mice: a new role for the RFRP receptor

Endocrinology ◽  
2021 ◽  
Author(s):  
Stephanie Constantin ◽  
Katherine Pizano ◽  
Kaya Matson ◽  
Yufei Shan ◽  
Daniel Reynolds ◽  
...  
Keyword(s):  
Neuropeptide Y ◽  
Fluorescent Protein ◽  
Nucleus Tractus Solitarius ◽  
Brain Slices ◽  
Adult Brain ◽  
Energy Deficit ◽  
Gonadotropin Secretion ◽  
Gnrh Neurons ◽  
Gonadotropin Inhibitory Hormone ◽  
Green Fluorescent

Abstract RFamide-related peptides (RFRPs, mammalian orthologs of gonadotropin-inhibitory hormone) convey circadian, seasonal and social cues to the reproductive system. They regulate gonadotropin secretion by modulating gonadotropin-releasing hormone (GnRH) neurons via the RFRP receptor. Mice lacking this receptor are fertile but exhibit abnormal gonadotropin responses during metabolic challenges such as acute fasting, when the normal drop in gonadotropin levels is delayed. Although it is known that these food intake signals to the reproductive circuit originate in the nucleus tractus solitarius (NTS) in the brainstem, the phenotype of the neurons conveying the signal remains unknown. Given that neuropeptide FF (NPFF), another RFamide peptide, resides in the NTS and can bind to the RFRP receptor, we hypothesized that NPFF may regulate GnRH neurons. To address this question, we used a combination of techniques: cell-attached electrophysiology on GnRH-driven green fluorescent protein-tagged neurons in acute brain slices; calcium imaging on cultured GnRH neurons; and immunostaining on adult brain tissue. We found 1) NPFF inhibits GnRH neuron excitability via the RFRP receptor and its canonical signaling pathway (Gi/o protein and G protein-coupled inwardly-rectifying potassium channels), 2) NPFF-like fibers in the vicinity of GnRH neurons coexpress neuropeptide Y, 3) the majority of NPFF-like cell bodies in the NTS also coexpress neuropeptide Y, and 4) acute fasting increased NPFF-like immunoreactivity in the NTS. Together these data indicate that NPFF neurons within the NTS inhibit GnRH neurons, and thus reproduction, during fasting but prior to the energy deficit.

scholarly journals Kiss1 Neurons Drastically Change Their Firing Activity in Accordance With the Reproductive State: Insights From a Seasonal Breeder

Endocrinology ◽  
2014 ◽  
Vol 155 (12) ◽  
pp. 4868-4880 ◽  
Author(s):  
Masaharu Hasebe ◽  
Shinji Kanda ◽  
Hiroyuki Shimada ◽  
Yasuhisa Akazome ◽  
Hideki Abe ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Resting Membrane Potential ◽  
Reproductive State ◽  
Firing Patterns ◽  
State Dependent ◽  
Channel Expression ◽  
Physiological Analysis ◽  
Seasonal Breeder ◽  
Green Fluorescent

Kisspeptin (Kiss) neurons show drastic changes in kisspeptin expression in response to the serum sex steroid concentration in various vertebrate species. Thus, according to the reproductive states, kisspeptin neurons are suggested to modulate various neuronal activities, including the regulation of GnRH neurons in mammals. However, despite their reproductive state-dependent regulation, there is no physiological analysis of kisspeptin neurons in seasonal breeders. Here we generated the first kiss1-enhanced green fluorescent protein transgenic line of a seasonal breeder, medaka, for histological and electrophysiological analyses using a whole-brain in vitro preparation in which most synaptic connections are intact. We found histologically that Kiss1 neurons in the nucleus ventralis tuberis (NVT) projected to the preoptic area, hypothalamus, pituitary, and ventral telencephalon. Therefore, NVT Kiss1 neurons may regulate various homeostatic functions and innate behaviors. Electrophysiological analyses revealed that they show various firing patterns, including bursting. Furthermore, we found that their firings are regulated by the resting membrane potential. However, bursting was not induced from the other firing patterns with a current injection, suggesting that it requires some chronic modulations of intrinsic properties such as channel expression. Finally, we found that NVT Kiss1 neurons drastically change their neuronal activities according to the reproductive state and the estradiol levels. Taken together with the previous reports, we here conclude that the breeding condition drastically alters the Kiss1 neuron activities in both gene expression and firing activities, the latter of which is strongly related to Kiss1 release, and the Kiss1 peptides regulate the activities of various neural circuits through their axonal projections.

scholarly journals 20-HETE-mediated cytotoxicity and apoptosis in ischemic kidney epithelial cells

2008 ◽  
Vol 294 (3) ◽  
pp. F562-F570 ◽  
Author(s):  
Vani Nilakantan ◽  
Cheryl Maenpaa ◽  
Guangfu Jia ◽  
Richard J. Roman ◽  
Frank Park
Keyword(s):  
Caspase 3 ◽  
Fluorescent Protein ◽  
Ischemia Reperfusion ◽  
Atp Depletion ◽  
Cellular Damage ◽  
Apoptotic Pathway ◽  
Injury Model ◽  
Green Fluorescent

20-HETE, a metabolite of arachidonic acid, has been implicated as a mediator of free radical formation and tissue death following ischemia-reperfusion (IR) injury in the brain and heart. The present study examined the role of this pathway in a simulated IR renal injury model in vitro. Modified self-inactivating lentiviral vectors were generated to stably overexpress murine Cyp4a12 following transduction into LLC-PK1 cells (LLC-Cyp4a12). We compared the survival of control and transduced LLC-PK1 cells following 4 h of ATP depletion and 2 h of recovery in serum-free medium. ATP depletion-recovery of LLC-Cyp4a12 cells resulted in a significantly higher LDH release ( P < 0.05) compared with LLC-enhanced green fluorescent protein (EGFP) cells. Treatment with the SOD mimetic MnTMPyP (100 μM) resulted in decreased cytotoxicity in LLC-Cyp4a12 cells. The selective 20-HETE inhibitor HET-0016 (10 μM) also inhibited cytotoxicity significantly ( P < 0.05) in LLC-Cyp4a12 cells. Dihydroethidium fluorescence showed that superoxide levels were increased to the same degree in LLC-EGFP and LLC-Cyp4a12 cells after ATP depletion-recovery compared with control cells and that this increase was inhibited by MnTMPyP. There was a significant increase ( P < 0.05) of caspase-3 cleavage, an effector protease of the apoptotic pathway, in the LLC-Cyp4a12 vs. LLC-EGFP cells ( P < 0.05). This was abolished in the presence of HET-0016 ( P < 0.05) or MnTMPyP ( P < 0.01). These results demonstrate that 20-HETE overexpression can significantly exacerbate the cellular damage that is associated with renal IR injury and that the programmed cell death is mediated by activation of caspase-3 and is partially dependent on enhanced CYP4A generation of free radicals.

Defining the Role of Arginine 96 in Green Fluorescent Protein Fluorophore Biosynthesis†,‡

Biochemistry ◽  
2005 ◽  
Vol 44 (49) ◽  
pp. 16211-16220 ◽  
Author(s):  
Timothy I. Wood ◽  
David P. Barondeau ◽  
Chiharu Hitomi ◽  
Carey J. Kassmann ◽  
John A. Tainer ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent

scholarly journals Targeting of Protein Kinase C-ϵ during Fcγ Receptor-dependent Phagocytosis Requires the ϵC1B Domain and Phospholipase C-γ1

2006 ◽  
Vol 17 (2) ◽  
pp. 799-813 ◽  
Author(s):  
Keylon L. Cheeseman ◽  
Takehiko Ueyama ◽  
Tanya M. Michaud ◽  
Kaori Kashiwagi ◽  
Demin Wang ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Phospholipase D ◽  
Fluorescent Protein ◽  
Wild Type ◽  
Fcγ Receptor ◽  
Kinase C ◽  
Green Fluorescent

Protein kinase C-ϵ (PKC-ϵ) translocates to phagosomes and promotes uptake of IgG-opsonized targets. To identify the regions responsible for this concentration, green fluorescent protein (GFP)-protein kinase C-ϵ mutants were tracked during phagocytosis and in response to exogenous lipids. Deletion of the diacylglycerol (DAG)-binding ϵC1 and ϵC1B domains, or the ϵC1B point mutant ϵC259G, decreased accumulation at phagosomes and membrane translocation in response to exogenous DAG. Quantitation of GFP revealed that ϵC259G, ϵC1, and ϵC1B accumulation at phagosomes was significantly less than that of intact PKC-ϵ. Also, the DAG antagonist 1-hexadecyl-2-acetyl glycerol (EI-150) blocked PKC-ϵ translocation. Thus, DAG binding to ϵC1B is necessary for PKC-ϵ translocation. The role of phospholipase D (PLD), phosphatidylinositol-specific phospholipase C (PI-PLC)-γ1, and PI-PLC-γ2 in PKC-ϵ accumulation was assessed. Although GFP-PLD2 localized to phagosomes and enhanced phagocytosis, PLD inhibition did not alter target ingestion or PKC-ϵ localization. In contrast, the PI-PLC inhibitor U73122 decreased both phagocytosis and PKC-ϵ accumulation. Although expression of PI-PLC-γ2 is higher than that of PI-PLC-γ1, PI-PLC-γ1 but not PI-PLC-γ2 consistently concentrated at phagosomes. Macrophages from PI-PLC-γ2-/-mice were similar to wild-type macrophages in their rate and extent of phagocytosis, their accumulation of PKC-ϵ at the phagosome, and their sensitivity to U73122. This implicates PI-PLC-γ1 as the enzyme that supports PKC-ϵ localization and phagocytosis. That PI-PLC-γ1 was transiently tyrosine phosphorylated in nascent phagosomes is consistent with this conclusion. Together, these results support a model in which PI-PLC-γ1 provides DAG that binds to ϵC1B, facilitating PKC-ϵ localization to phagosomes for efficient IgG-mediated phagocytosis.

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