scholarly journals Dose-Dependent Switch in Response of Gonadotropin-Releasing Hormone (GnRH) Neurons to GnRH Mediated through the Type I GnRH Receptor

Endocrinology ◽  
2004 ◽  
Vol 145 (2) ◽  
pp. 728-735 ◽  
Author(s):  
Chun Xu ◽  
Xu-Zhi Xu ◽  
Craig S. Nunemaker ◽  
Suzanne M. Moenter
Keyword(s):  
Firing Rate ◽  
Fluorescent Protein ◽  
Brain Slices ◽  
Dose Dependence ◽  
Gnrh Receptor ◽  
Neuron Activity ◽  
Type I ◽  
Pulsatile Release ◽  
Gnrh Neurons ◽  
Green Fluorescent

Abstract Pulsatile release of GnRH provides central control of reproduction. GnRH neuron activity is likely synchronized to produce hormone pulses, but the mechanisms are largely unknown. One candidate for communication among these neurons is GnRH itself. Cultured embryonic and immortalized GnRH neurons express GnRH receptor type I (GnRHR-1), but expression has not been shown in adult GnRH neurons. Using mice that express green fluorescent protein (GFP) in GnRH neurons, we tested whether adult GnRH neurons express GnRHR-1. GFP-positive (n = 42) and -negative neurons (n = 22) were harvested from brain slices, and single-cell RT-PCR was performed with cell contents. Fifty-two percent of the GnRH neurons tested expressed GnRHR-1, but only 9% of non-GnRH hypothalamic neurons expressed GnRHR-1; no false harvest controls (n = 13) were positive. GnRHR-1 expression within GnRH neurons suggested a physiological ultrashort loop feedback role for GnRH. Thus, we examined the effect of GnRH on the firing rate of GnRH neurons. Low-dose GnRH (20 nm) significantly decreased firing rate in 12 of 22 neurons (by 42 ± 4%, P < 0.05), whereas higher doses increased firing rate (200 nm, five of 10 neurons, 72 ± 26%; 2000 nm, nine of 13 neurons, 53 ± 8%). Interestingly, the fraction of GnRH neurons responding was similar to the fraction in which GnRHR-1 was detected. Together, these data demonstrate that a subpopulation of GnRH neurons express GnRHR-1 and respond to GnRH with altered firing. The dose dependence suggests that this autocrine control of GnRH neurons may be not only a mechanism for generating and modulating pulsatile release, but it may also be involved in the switch between pulse and surge modes of release.

scholarly journals RFamide-Related Peptide-3, a Mammalian Gonadotropin-Inhibitory Hormone Ortholog, Regulates Gonadotropin-Releasing Hormone Neuron Firing in the Mouse

Endocrinology ◽  
2009 ◽  
Vol 150 (6) ◽  
pp. 2799-2804 ◽  
Author(s):  
Eric Ducret ◽  
Greg M. Anderson ◽  
Allan E. Herbison
Keyword(s):  
Firing Rate ◽  
Fluorescent Protein ◽  
Suppressive Effect ◽  
Small Population ◽  
Neuron Activity ◽  
Gonadotropin Secretion ◽  
Gnrh Neurons ◽  
Gonadotropin Inhibitory Hormone ◽  
Green Fluorescent ◽  
Prolactin Releasing Peptide

The recent discovery that an RFamide termed gonadotropin-inhibitory hormone is likely to be a hypophysiotrophic gonadotropin release-inhibiting hormone in birds has generated interest into the role of LPXRFamide neuropeptides in the control of gonadotropin secretion in mammals. Recent immunocytochemical studies in birds and mammals have suggested that neurons expressing the mammalian LPXRFamides, RFamide-related peptides (RFRPs) 1 and 3, may innervate and regulate GnRH neurons directly. We used cell-attached electrophysiology in adult male and female GnRH-green fluorescent protein-tagged neurons to examine whether RFRP-3 modulated the electrical excitability of GnRH neurons. RFRP-3 was found to exhibit rapid and repeatable inhibitory effects on the firing rate of 41% of GnRH neurons. A small population of GnRH neurons (12%) increased their firing rate in response to RFRP-3, and the remainder was unaffected. No difference was detected in the RFRP-3 responses of GnRH neurons from male, diestrous, or proestrus female mice. The suppressive effect of RFRP-3 was maintained when amino acid transmission was blocked, suggesting a possible direct effect of RFRP-3 upon GnRH neurons. To evaluate the effects of other RFamide neuropeptides on GnRH neurons, we tested the actions of prolactin-releasing peptide-20 and -31. Neither compounds altered the firing rate of GnRH neurons. These studies demonstrate that RFRP-3 has a likely direct suppressive action on the excitability of GnRH neurons, indicating a role for RFRPs in the regulation of gonadotropin secretion in mammals through modulation of GnRH neuron activity.

scholarly journals Neurosteroids Alter γ-Aminobutyric Acid Postsynaptic Currents in Gonadotropin-Releasing Hormone Neurons: A Possible Mechanism for Direct Steroidal Control

Endocrinology ◽  
2003 ◽  
Vol 144 (10) ◽  
pp. 4366-4375 ◽  
Author(s):  
Shannon D. Sullivan ◽  
Suzanne M. Moenter
Keyword(s):  
Gabaa Receptor ◽  
Decay Time ◽  
Chloride Channels ◽  
Fluorescent Protein ◽  
Brain Slices ◽  
Cell Voltage ◽  
Dehydroepiandrosterone Sulfate ◽  
Postsynaptic Currents ◽  
Gnrh Neurons ◽  
Green Fluorescent

Pulsatile GnRH release is required for fertility and is regulated by steroid feedback. Whether or not steroids or their metabolites act directly on GnRH neurons is not well established. In some neurons, steroid metabolites known as neurosteroids modulate the function of the GABAA receptor. Specifically, the progesterone derivative allopregnanolone is an allosteric agonist at this receptor, whereas the androgen dehydroepiandrosterone sulfate (DHEAS) is an allosteric antagonist. We hypothesized these metabolites act similarly on GnRH neurons to modify the response to GABA. Whole-cell voltage-clamp recordings of GABAergic miniature postsynaptic currents (mPSCs) were made from green fluorescent protein-identified GnRH neurons in brain slices from diestrous mice. Glutamatergic currents were blocked with antagonists and action potentials blocked with tetrodotoxin, minimizing presynaptic effects of treatments. Allopregnanolone (5 μm) increased mPSC rate of rise, amplitude and decay time by 15.9 ± 6.1%, 16.5 ± 6.3%, and 58.3 ± 18.6%, respectively (n = 7 cells). DHEAS (5 μm) reduced mPSC rate of rise (32.1 ± 5.7%) and amplitude (27.6 ± 4.3%) but did not alter decay time (n = 8). Effects of both neurosteroids were dose dependent between 0.1 and 10 μm. In addition to independent actions, DHEAS also reversed effects of allopregnanolone on rate of rise and amplitude so that these parameters were returned to pretreatment baseline values (n = 6). These data indicate allopregnanolone increases and DHEAS decreases responsiveness of GnRH neurons to activation of GABAA receptors by differentially modulating current flow through GABAA receptor chloride channels. This provides one mechanism for direct steroid feedback to GnRH neurons.

scholarly journals Ovarian Androgens Maintain High GnRH Neuron Firing Rate in Adult Prenatally-Androgenized Female Mice

Endocrinology ◽  
2019 ◽  
Vol 161 (1) ◽  
Author(s):  
Eden A Dulka ◽  
Laura L Burger ◽  
Suzanne M Moenter
Keyword(s):  
Firing Rate ◽  
Fluorescent Protein ◽  
Polycystic Ovary ◽  
Neuron Activity ◽  
Gnrh Neuron ◽  
Neuron Firing ◽  
Dependent Change ◽  
Gnrh Release ◽  
Green Fluorescent ◽  
Reproductive Cycles

Abstract Changes in gonadotropin-releasing hormone (GnRH) release frequency from the brain help drive reproductive cycles. In polycystic ovary syndrome (PCOS), persistent high GnRH/luteinizing hormone (LH) frequency disrupts cycles and exacerbates hyperandrogenemia. Adult prenatally-androgenized (PNA) mice exhibit increased GnRH neuron firing rate, elevated ovarian androgens, and disrupted cycles, but before puberty, GnRH neuron activity is reduced in PNA mice compared with controls. We hypothesized that ovarian feedback mediates the age-dependent change in GnRH neuron firing rate in PNA vs control mice. Extracellular recordings of green fluorescent protein (GFP)-identified GnRH neurons were made 5 to 7 days after sham-surgery, ovariectomy (OVX), or, in adults, after OVX plus replacement of sub-male androgen levels with dihydrotestosterone implants (OVX + DHT). In 3-week-old mice, OVX did not affect GnRH neuron firing rate in either group. In adult controls, OVX increased GnRH neuron firing rate, which was further enhanced by DHT. In adult PNA mice, however, OVX decreased GnRH neuron firing rate, and DHT restored firing rate to sham-operated levels. In contrast to the differential effects of ovarian feedback on GnRH neuron firing rate, serum LH increased after OVX in both control and PNA mice and was not altered by DHT. Pituitary gene expression largely reflected changes expected with OVX, although in PNA but not control mice, DHT treatment increased Lhb expression. These results suggest prenatal androgen exposure programs marked changes in GnRH neuron regulation by homeostatic steroid feedback. PNA lowers GnRH neuron activity in low-steroid states (before puberty, OVX), and renders activity in adulthood dependent upon ongoing exposure to elevated ovarian androgens.

scholarly journals Androgens Increase Gonadotropin-Releasing Hormone Neuron Firing Activity in Females and Interfere with Progesterone Negative Feedback

Endocrinology ◽  
2006 ◽  
Vol 147 (3) ◽  
pp. 1474-1479 ◽  
Author(s):  
Justyna Pielecka ◽  
Samuel D. Quaynor ◽  
Suzanne M. Moenter
Keyword(s):  
Animal Models ◽  
Firing Rate ◽  
Negative Feedback ◽  
Brain Slices ◽  
Neuron Activity ◽  
Gnrh Neuron ◽  
Firing Activity ◽  
Neuron Firing ◽  
Gnrh Neurons ◽  
Gaba Transmission

GnRH neurons are the central regulators of fertility, and their activity is modulated by steroid feedback. In women with hyperandrogenemic infertility and in animal models of these disorders, elevated androgen levels interfere with progesterone (P) negative feedback. Our previous work showed that steroids altered the frequency and amplitude of γ-aminobutyric acid (GABA) transmission to GnRH neurons. Specifically, P inhibited GABA transmission, which can excite GnRH neurons, whereas dihydrotestosterone (DHT) increased GABA transmission. In this study the GnRH neuron firing rate was examined in the same animal models. Adult (>2 months) female mice were ovariectomized and treated for 8–12 d with implants containing estradiol (E), E and P, E and DHT, or E, P, and DHT. Targeted extracellular recordings were used to examine the long-term firing activity of green fluorescent protein-identified GnRH neurons in brain slices from these mice. In comparing E alone to E plus P animals, P increased the percentage of time that GnRH neurons were quiescent and reduced the area under the curve of the firing rate and the instantaneous firing frequency, suggesting that P provides additional negative feedback over E alone. The addition of DHT markedly increased GnRH neuron activity in both the presence and absence of P. DHT also altered the firing pattern of GnRH neurons, such that peaks in the firing rate detected by the Cluster8 algorithm were approximately doubled in frequency and amplitude. These data support and extend our previous findings and are consistent with the hypothesis that the changes in GABAergic transmission observed in these animal models impact upon the activity of GnRH neurons, and central androgen action probably stimulates GnRH release.

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 The Regularity of Sustained Firing Reveals Two Populations of Slowly Adapting Touch Receptors in Mouse Hairy Skin

2010 ◽  
Vol 103 (6) ◽  
pp. 3378-3388 ◽  
Author(s):  
Scott A. Wellnitz ◽  
Daine R. Lesniak ◽  
Gregory J. Gerling ◽  
Ellen A. Lumpkin
Keyword(s):  
Fluorescent Protein ◽  
Ex Vivo ◽  
Receptive Fields ◽  
Receptor Subtypes ◽  
Type I ◽  
Genetic Dissection ◽  
Merkel Cells ◽  
Simple Method ◽  
Green Fluorescent ◽  
Nerve Recordings

Touch is initiated by diverse somatosensory afferents that innervate the skin. The ability to manipulate and classify receptor subtypes is prerequisite for elucidating sensory mechanisms. Merkel cell–neurite complexes, which distinguish shapes and textures, are experimentally tractable mammalian touch receptors that mediate slowly adapting type I (SAI) responses. The assessment of SAI function in mutant mice has been hindered because previous studies did not distinguish SAI responses from slowly adapting type II (SAII) responses, which are thought to arise from different end organs, such as Ruffini endings. Thus we sought methods to discriminate these afferent types. We developed an epidermis-up ex vivo skin–nerve chamber to record action potentials from afferents while imaging Merkel cells in intact receptive fields. Using model-based cluster analysis, we found that two types of slowly adapting receptors were readily distinguished based on the regularity of touch-evoked firing patterns. We identified these clusters as SAI (coefficient of variation = 0.78 ± 0.09) and SAII responses (0.21 ± 0.09). The identity of SAI afferents was confirmed by recording from transgenic mice with green fluorescent protein–expressing Merkel cells. SAI receptive fields always contained fluorescent Merkel cells ( n = 10), whereas SAII receptive fields lacked these cells ( n = 5). Consistent with reports from other vertebrates, mouse SAI and SAII responses arise from afferents exhibiting similar conduction velocities, receptive field sizes, mechanical thresholds, and firing rates. These results demonstrate that mice, like other vertebrates, have two classes of slowly adapting light-touch receptors, identify a simple method to distinguish these populations, and extend the utility of skin–nerve recordings for genetic dissection of touch receptor mechanisms.

scholarly journals Vaccinia Virus F13L Protein with a Conserved Phospholipase Catalytic Motif Induces Colocalization of the B5R Envelope Glycoprotein in Post-Golgi Vesicles

2001 ◽  
Vol 75 (16) ◽  
pp. 7528-7542 ◽  
Author(s):  
Matloob Husain ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Fluorescent Protein ◽  
Plasmid Vector ◽  
Recombinant Vaccinia Virus ◽  
Open Reading Frames ◽  
Type I ◽  
Golgi Vesicles ◽  
Infected Cells ◽  
Catalytic Motif ◽  
Green Fluorescent

ABSTRACT The wrapping of intracellular mature vaccinia virions by modifiedtrans-Golgi or endosomal cisternae to form intracellular enveloped virions is dependent on at least two viral proteins encoded by the B5R and F13L open reading frames. B5R is a type I integral membrane glycoprotein, whereas F13L is an unglycosylated, palmitylated protein with a motif that is conserved in a superfamily of phospholipid-metabolizing enzymes. Microscopic visualization of the F13L protein was achieved by fusing it to the enhanced green fluorescent protein (GFP). F13L-GFP was functional when expressed by a recombinant vaccinia virus in which it replaced the wild-type F13L gene or by transfection of uninfected cells with a plasmid vector followed by infection with an F13L deletion mutant. In uninfected or infected cells, F13L-GFP was associated with Golgi cisternae and post-Golgi vesicles containing the LAMP 2 late endosomal-lysosomal marker. Association of F13L-GFP with vesicles was dependent on an intact phospholipase catalytic motif and sites of palmitylation. The B5R protein was also associated with LAMP2-containing vesicles when F13L-GFP was coexpressed, but was largely restricted to Golgi cisternae in the absence of F13L-GFP or when the F13L moiety was mutated. We suggest that the F13L protein, like its human phospholipase D homolog, regulates vesicle formation and that this process is involved in intracellular enveloped virion membrane formation.

scholarly journals Effects of acute and chronic nicotine on catecholamine neurons of the nucleus of the solitary tract

2019 ◽  
Vol 316 (1) ◽  
pp. R38-R49
Author(s):  
Stephen J. Page ◽  
Mingyan Zhu ◽  
Suzanne M. Appleyard
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Fluorescent Protein ◽  
Brain Slices ◽  
Nicotine Withdrawal ◽  
Cardiovascular Reflexes ◽  
Solitary Tract ◽  
Catecholamine Neurons ◽  
Green Fluorescent ◽  
Tyrosine Hydroxylase Promoter

Nicotine is an addictive drug that has broad effects throughout the brain. One site of action is the nucleus of the solitary tract (NTS), where nicotine initiates a stress response and modulates cardiovascular and gastric function through nicotinic acetylcholine receptors (nAChRs). Catecholamine (CA) neurons in the NTS influence stress and gastric and cardiovascular reflexes, making them potential mediators of nicotine’s effects; however nicotine’s effect on these neurons is unknown. Here, we determined nicotine’s actions on NTS-CA neurons by use of patch-clamp techniques in brain slices from transgenic mice expressing enhanced green fluorescent protein driven by the tyrosine hydroxylase promoter (TH-EGFP). Picospritzing nicotine both induced a direct inward current and increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in NTS-CA neurons, effects blocked by nonselective nAChR antagonists TMPH and MLA. The increase in sEPSC frequency was mimicked by nAChRα7 agonist AR-R17779 and blocked by nAChRα7 antagonist MG624. AR-R17779 also increased the firing of TH-EGFP neurons, an effect dependent on glutamate inputs, as it was blocked by the glutamate antagonist NBQX. In contrast, the nicotine-induced current was mimicked by nAChRα4β2 agonist RJR2403 and blocked by nAChRα4β2 antagonist DHβE. RJR2403 also increased the firing rate of TH-EGFP neurons independently of glutamate. Finally, both somatodendritic and sEPSC nicotine responses from NTS-CA neurons were larger in nicotine-dependent mice that had under gone spontaneous nicotine withdrawal. These results demonstrate that 1) nicotine activates NTS-CA neurons both directly, by inducing a direct current, and indirectly, by increasing glutamate inputs, and 2) NTS-CA nicotine responsiveness is altered during nicotine withdrawal.

Interleukin-1 (IL-1) Inhibits Growth of Cytomegalovirus in Human Marrow Stromal Cells: Inhibition Is Reversed Upon Removal of IL-1

Blood ◽  
1999 ◽  
Vol 94 (2) ◽  
pp. 572-578 ◽  
Author(s):  
Mineo Iwata ◽  
Jeff Vieira ◽  
Michael Byrne ◽  
Heidi Horton ◽  
Beverly Torok-Storb
Keyword(s):  
Stromal Cells ◽  
Neutralizing Antibodies ◽  
Fluorescent Protein ◽  
Interleukin 1 ◽  
Elongation Factor ◽  
Marrow Stromal Cells ◽  
Type I ◽  
Untreated Culture ◽  
Green Fluorescent ◽  
Induced Changes

Abstract A Toledo strain cytomegalovirus (CMV) containing the gene for green fluorescent protein (GFP) under the control of elongation factor-1 promoter was used to study infection of human marrow stromal cells. Two stromal cell lines were used: HS-5, which secretes copious amounts of known cytokines and interleukins; and HS-27a, which does not secrete these activities. CMV growth and spread was monitored by counting green plaques and quantitating GFP intensity. Initial studies indicated that, whereas HS-5 and 27a have similar susceptibilities to infection, as evidenced by the same number of GFP+ cells at day 2, HS-5 appears more resistant to growth and spread of CMV. Furthermore, conditioned media from HS-5 (HS-5 CM) inhibited CMV plaque formation in HS-27a, suggesting that factors secreted by HS-5 are responsible for limiting CMV growth. Neutralizing antibodies against interleukin-1 (IL-1) and IL-1β completely blocked the ability of HS-5 CM to limit viral growth, suggesting that IL-1, which is known to be present in HS-5 CM, is responsible for this effect. When exogenous IL-1β was added to CMV-infected HS-27a, both the number of plaques and the intensity of GFP was significantly reduced in IL-1–treated HS-27a compared with untreated HS-27a (the number of plaques by day 18 was 20 ± 3 v 151 ± 12/well, respectively; GFP intensity was 535 ± 165 v 6,516 ± 652/well, respectively, in 4 separate experiments). At day 21, when IL-1β–treated, CMV-infected cultures were passaged and then cultured in the absence of IL-1β, CMV growth progressed with the kinetics of the original untreated culture, indicating that the IL-1β effect is reversible. Because HS-27a expresses the type I IL-1 receptor, we speculate that the antiviral effects are mediated through IL-1–induced changes in cellular gene expression. DNA chip analysis of mRNA from IL-1β–treated and nontreated HS-27a cells has identified some candidate molecules.

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