Lack of the Kir4.1 Channel Subunit Abolishes K+ Buffering Properties of Astrocytes in the Ventral Respiratory Group: Impact on Extracellular K+ Regulation

2006 ◽  
Vol 95 (3) ◽  
pp. 1843-1852 ◽  
Author(s):  
Clemens Neusch ◽  
Nestoras Papadopoulos ◽  
Michael Müller ◽  
Iris Maletzki ◽  
Stefan M. Winter ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Respiratory Rhythm ◽  
Brain Slices ◽  
Physiological Role ◽  
Functional Expression ◽  
Network Activity ◽  
Early Postnatal Development ◽  
Ventral Respiratory Group ◽  
Respiratory Network ◽  
Green Fluorescent

Ongoing rhythmic neuronal activity in the ventral respiratory group (VRG) of the brain stem results in periodic changes of extracellular K+. To estimate the involvement of the weakly inwardly rectifying K+ channel Kir4.1 (KCNJ10) in extracellular K+ clearance, we examined its functional expression in astrocytes of the respiratory network. Kir4.1 was expressed in astroglial cells of the VRG, predominantly in fine astrocytic processes surrounding capillaries and in close proximity to VRG neurons. Kir4.1 expression was up-regulated during early postnatal development. The physiological role of astrocytic Kir4.1 was studied using mice with a null mutation in the Kir4.1 channel gene that were interbred with transgenic mice expressing the enhanced green fluorescent protein in their astrocytes. The membrane potential was depolarized in astrocytes of Kir4.1−/− mice, and Ba2+-sensitive inward K+ currents were diminished. Brain slices from Kir4.1−/− mice, containing the pre-Bötzinger complex, which generates a respiratory rhythm, did not show any obvious differences in rhythmic bursting activity compared with wild-type controls, indicating that the lack of Kir4.1 channels alone does not impair respiratory network activity. Extracellular K+ measurements revealed that Kir4.1 channels contribute to extracellular K+ regulation. Kir4.1 channels reduce baseline K+ levels, and they compensate for the K+ undershoot. Our data indicate that Kir4.1 channels 1) are expressed in perineuronal processes of astrocytes, 2) constitute the major part of the astrocytic Kir conductance, and 3) contribute to regulation of extracellular K+ in the respiratory network.

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.

Activity of the renal Na+-K+-2Cl− cotransporter is reduced by mutagenesis of N-glycosylation sites: role for protein surface charge in Cl− transport

2006 ◽  
Vol 290 (5) ◽  
pp. F1094-F1102 ◽  
Author(s):  
Anahí Paredes ◽  
Consuelo Plata ◽  
Manuel Rivera ◽  
Erika Moreno ◽  
Norma Vázquez ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Fluorescent Protein ◽  
Functional Expression ◽  
Site Directed Mutagenesis ◽  
Intrinsic Activity ◽  
Xenopus Laevis Oocytes ◽  
Type I ◽  
Wild Type ◽  
Glycosylation Sites ◽  
Green Fluorescent

The renal-specific Na+-K+-2Cl− cotransporter NKCC2 belongs to the SLC12 gene family; it is the target for loop diuretics and the cause of type I Bartter's syndrome. Because the NKCC2 sequence contains two putative N-linked glycosylation sites, one of which is conserved with the renal Na+-Cl− cotransporter in which glycosylation affects thiazide affinity, we assessed the role of glycosylation on NKCC2 functional properties. One (N442Q or N452Q) or both (N442,452Q) N-glycosylation sites were eliminated by site-directed mutagenesis. Wild-type NKCC2 and mutant clones were expressed in Xenopus laevis oocytes and analyzed by 86Rb+ influx, Western blotting, and confocal microscopy. Inhibition of glycosylation with tunicamycin in wild-type NKCC2-injected oocytes resulted in an 80% reduction of NKCC2 activity. Immunoblot of injected oocytes revealed that glycosylation of NKCC2 was completely prevented in N442,452Q-injected oocytes. Functional activity was reduced by 50% in N442Q- and N452Q-injected oocytes and by 80% in oocytes injected with N442,452Q, whereas confocal microscopy of oocytes injected with wild-type or mutant enhanced green fluorescent protein-tagged NKCC2 clones revealed that surface fluorescence intensity was reduced ∼20% in single mutants and 50% in the double mutant. Ion transport kinetic analyses revealed no changes in cation affinity and a small increase in Cl− affinity by N442Q and N442,452Q. However, a slight decrease in bumetanide affinity was observed. Our data demonstrate that NKCC2 is glycosylated and suggest that prevention of glycosylation reduces its functional expression by affecting insertion into the plasma membrane and the intrinsic activity of the cotransporter.

scholarly journals Nuclear targeting of the β isoform of Type II phosphatidylinositol phosphate kinase (phosphatidylinositol 5-phosphate 4-kinase) by its α-helix 7

2000 ◽  
Vol 346 (3) ◽  
pp. 587-591 ◽  
Author(s):  
Antonio CIRUELA ◽  
Katherine A. HINCHLIFFE ◽  
Nullin DIVECHA ◽  
Robin F. IRVINE
Keyword(s):  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Physiological Role ◽  
Nuclear Localization Sequence ◽  
Type Ii ◽  
Nuclear Targeting ◽  
Phosphatidylinositol Phosphate ◽  
Localization Sequence ◽  
Α Helix ◽  
Green Fluorescent

Type II phosphatidylinositol phosphate kinases (PIPkins) have recently been found to be primarily phosphatidylinositol 5-phosphate 4-kinases, and their physiological role remains unclear. We have previously shown that a Type II PIPkin [isoform(s) unknown], is localized partly in the nucleus [Divecha, Rhee, Letcher and Irvine (1993) Biochem. J. 289, 617-620], and here we show, by transfection of HeLa cells with green-fluorescent-protein-tagged Type II PIPkins, that this is likely to be the Type IIβ isoform. Type IIβ PIPkin has no obvious nuclear localization sequence, and a detailed analysis of the localization of chimaeras and mutants of the α (cytosolic) and β PIPkins shows that the nuclear localization requires the presence of a 17-amino-acid length of α-helix (α-helix 7) that is specific to the β isoform, and that this helix must be present in its entirety, with a precise orientation. This resembles the nuclear targeting of the HIV protein Vpr, and Type IIβ PIPkin is apparently therefore the first example of a eukaryotic protein that uses the same mechanism.

scholarly journals Activation of Neurokinin 3 Receptors Stimulates GnRH Release in a Location-Dependent but Kisspeptin-Independent Manner in Adult Mice

Endocrinology ◽  
2013 ◽  
Vol 154 (11) ◽  
pp. 3984-3989 ◽  
Author(s):  
Garrett T. Gaskins ◽  
Katarzyna M. Glanowska ◽  
Suzanne M. Moenter
Keyword(s):  
Green Fluorescent Protein ◽  
Carbon Fiber ◽  
Fluorescent Protein ◽  
Hypogonadotropic Hypogonadism ◽  
Brain Slices ◽  
Dependent Manner ◽  
Gnrh Secretion ◽  
Gnrh Release ◽  
Carbon Fiber Microelectrodes ◽  
Green Fluorescent

GnRH neurons form the final common pathway for the central control of reproduction. GnRH release occurs from terminals in the external layer of the median eminence (ME) for neuroendocrine control of the pituitary, and near GnRH-GnRH fiber appositions within the preoptic area (POA). Whether or not control of GnRH secretion by neuromodulators is different in these 2 areas is unknown. Mutations in neurokinin B (NKB) or the neurokinin-3 receptor (NK3R) are linked to hypogonadotropic hypogonadism in humans, suggesting that NKB may regulate GnRH secretion. Using fast scan cyclic voltammetry through carbon-fiber microelectrodes, we examined real-time GnRH release in response to the NK3R agonist senktide in the ME and POA. Coronal brain slices were acutely prepared from adult gonad-intact GnRH-green fluorescent protein male mice, and carbon-fiber microelectrodes were placed either within green fluorescent protein-positive terminal fields of the ME or near GnRH-GnRH fiber appositions in the POA. Senktide induced GnRH release consistently in the ME but not the POA, indicating that GnRH release is differentially regulated by NKB in a location-dependent manner. Senktide also induced GnRH secretion in the ME of kisspeptin-knockout (Kiss1 knockout) mice. Interestingly, release amplitude was lower compared with wild-type mice. These data indicate regulation of GnRH release by NK3R agonists is site specific and suggest that kisspeptin is not a required mediator between NK3R activation and GnRH secretion in the ME. This information will be useful for informing future models of afferent regulation of GnRH release.

scholarly journals Aminophylline modulation of the mouse respiratory network changes during postnatal maturation

2000 ◽  
Vol 89 (5) ◽  
pp. 2015-2022 ◽  
Author(s):  
B. Wilken ◽  
J. M. Ramirez ◽  
F. Hanefeld ◽  
D. W. Richter
Keyword(s):  
Developmental Change ◽  
Respiratory Rhythm ◽  
Respiratory Control ◽  
Network Activity ◽  
Signal Pathways ◽  
Control Mechanisms ◽  
Patch Clamp Technique ◽  
Postnatal Maturation ◽  
Respiratory Network ◽  
Synaptic Drive

Aminophylline is a respiratory stimulant commonly used for the treatment of central apnea. Experiences from clinical practice, however, revealed that aminophylline is not reliably effective in preterm infants, whereas it is normally effective in infants and mature patients. In an established animal model for postnatal development of respiratory control mechanisms, we therefore examined the hypothesis that the clinical observations reflect a developmental change in the sensitivity of the central respiratory network to methylxanthines. The medullary respiratory network was isolated at different postnatal ages ( postnatal days 1–13; P1–P13) in a transverse mouse brain stem slice preparation. This preparation contains the pre-Bötzinger complex (PBC), a region that is critical for generation of respiratory rhythm. Spontaneous rhythmic respiratory activity was recorded from the hypoglossal (XII) rootlets and from neurons in the PBC by using the whole cell patch clamp technique. Bath-applied aminophylline [20 μM] increased the frequency (+41%) in neonatal animals (P1–P6) without affecting the amplitude of respiratory burst activity in XII rootlets. The same concentration of aminophylline did not have any significant effect on the frequency of respiratory XII bursts but increased the amplitude (+31%) in juvenile animals (P7–P13). In the same age group, aminophylline also augmented the amplitude and the duration of respiratory synaptic drive currents in respiratory PBC neurons. The data demonstrate that augmentation of the respiratory output is due to direct enhancement of central respiratory network activity and increase of synaptic drive of hypoglossal motoneurons in juvenile, but not neonatal, animals. This indicates a developmental change in the efficacy of aminophylline to reinforce central respiratory network activity. Therefore, we believe that the variable success in treating respiratory disturbances in premature infants reflects maturational changes in the expression of receptors and/or intracellular signal pathways in the central respiratory network.

scholarly journals Characterization of Leptin-Responsive Neurons in the Caudal Brainstem

Endocrinology ◽  
2006 ◽  
Vol 147 (7) ◽  
pp. 3190-3195 ◽  
Author(s):  
Kate L. J. Ellacott ◽  
Ilia G. Halatchev ◽  
Roger D. Cone
Keyword(s):  
Green Fluorescent Protein ◽  
Energy Homeostasis ◽  
Leptin Receptor ◽  
Fluorescent Protein ◽  
Physiological Role ◽  
Cell Group ◽  
Melanocortin System ◽  
Peptide Precursor ◽  
Green Fluorescent

The central melanocortin system plays a key role in the regulation of energy homeostasis. Neurons containing the peptide precursor proopiomelanocortin (POMC) are found at two sites in the brain, the arcuate nucleus of the hypothalamus (ARC) and the caudal region of the nucleus of the solitary tract (NTS). ARC POMC neurons, which also express cocaine- and amphetamine-regulated transcript (CART), are known to mediate part of the response to factors regulating energy homeostasis, such as leptin and ghrelin. In contrast, the physiological role(s) of the POMC neurons in the caudal brainstem are not well characterized. However, development of a transgenic mouse expressing green fluorescent protein under the control of the POMC promoter [POMC-enhanced green fluorescent protein (EGFP) mouse] has aided the study of these neurons. Indeed, recent studies have shown significant activation of NTS POMC-EGFP cells by the gut released satiety factor cholecystokinin (CCK). Here we show that peripheral leptin administration induces the expression of phospho-signal transducer and activator of transcription 3 immunoreactivity (pSTAT3-IR), a marker of leptin receptor signaling, in more than 50% of NTS POMC-EGFP neurons. Furthermore, these POMC-EGFP neurons comprise 30% of all pSTAT3-IR cells in the NTS. Additionally, we also show that in contrast to the ARC population, NTS POMC-EGFP neurons do not coexpress CART immunoreactivity. These data suggest that NTS POMC neurons may participate with ARC POMC cells in mediating some of the effects of leptin and thus comprise a novel cell group regulated by both long-term adipostatic signals and satiety factors such as CCK.

scholarly journals Imaging of respiratory-related population activity with single-cell resolution

2007 ◽  
Vol 292 (1) ◽  
pp. C508-C516 ◽  
Author(s):  
Frank Funke ◽  
Mathias Dutschmann ◽  
Michael Müller
Keyword(s):  
Single Cell ◽  
Neuronal Activity ◽  
Single Cells ◽  
Activity Patterns ◽  
Respiratory Rhythm ◽  
Network Activity ◽  
Respiratory Neurons ◽  
Ventrolateral Medulla ◽  
Population Activity ◽  
Respiratory Network

The pre-Bötzinger complex (PBC) in the rostral ventrolateral medulla contains a kernel involved in respiratory rhythm generation. So far, its respiratory activity has been analyzed predominantly by electrophysiological approaches. Recent advances in fluorescence imaging now allow for the visualization of neuronal population activity in rhythmogenic networks. In the respiratory network, voltage-sensitive dyes have been used mainly, so far, but their low sensitivity prevents an analysis of activity patterns of single neurons during rhythmogenesis. We now have succeeded in using more sensitive Ca2+ imaging to study respiratory neurons in rhythmically active brain stem slices of neonatal rats. For the visualization of neuronal activity, fluo-3 was suited best in terms of neuronal specificity, minimized background fluorescence, and response magnitude. The tissue penetration of fluo-3 was improved by hyperosmolar treatment (100 mM mannitol) during dye loading. Rhythmic population activity was imaged with single-cell resolution using a sensitive charge-coupled device camera and a ×20 objective, and it was correlated with extracellularly recorded mass activity of the contralateral PBC. Correlated optical neuronal activity was obvious online in 29% of slices. Rhythmic neurons located deeper became detectable during offline image processing. Based on their activity patterns, 74% of rhythmic neurons were classified as inspiratory and 26% as expiratory neurons. Our approach is well suited to visualize and correlate the activity of several single cells with respiratory network activity. We demonstrate that neuronal synchronization and possibly even network configurations can be analyzed in a noninvasive approach with single-cell resolution and at frame rates currently not reached by most scanning-based imaging techniques.

scholarly journals Functional properties of human ferroportin, a cellular iron exporter reactive also with cobalt and zinc

2014 ◽  
Vol 306 (5) ◽  
pp. C450-C459 ◽  
Author(s):  
Colin J. Mitchell ◽  
Ali Shawki ◽  
Tomas Ganz ◽  
Elizabeta Nemeth ◽  
Bryan Mackenzie
Keyword(s):  
Iron Homeostasis ◽  
Fluorescent Protein ◽  
Serum Zinc ◽  
Functional Expression ◽  
Extracellular Ph ◽  
Zinc Homeostasis ◽  
Affinity Constant ◽  
Protein Fusion ◽  
Zinc Levels ◽  
Green Fluorescent

Iron homeostasis is achieved by regulating the intestinal absorption of the metal and its recycling by macrophages. Iron export from enterocytes or macrophages to blood plasma is thought to be mediated by ferroportin under the control of hepcidin. Although ferroportin was identified over a decade ago, little is understood about how it works. We expressed in Xenopus oocytes a human ferroportin-enhanced green fluorescent protein fusion protein and observed using confocal microscopy its exclusive plasma-membrane localization. As a first step in its characterization, we established an assay to detect functional expression of ferroportin by microinjecting oocytes with 55Fe and measuring efflux. Ferroportin expression increased the first-order rate constants describing 55Fe efflux up to 300-fold over control. Ferroportin-mediated 55Fe efflux was saturable, temperature-dependent (activation energy, Ea ≈ 17 kcal/mol), maximal at extracellular pH ≈ 7.5, and inactivated at extracellular pH < 6.0. We estimated that ferroportin reacts with iron at its intracellular aspect with apparent affinity constant < 10−7 M. Ferroportin expression also stimulated efflux of 65Zn and 57Co but not of 64Cu, 109Cd, or 54Mn. Hepcidin treatment of oocytes inhibited efflux of 55Fe, 65Zn, and 57Co. Whereas hepcidin administration in mice resulted in a marked hypoferremia within 4 h, we observed no effect on serum zinc levels in those same animals. We conclude that ferroportin is an iron-preferring cellular metal-efflux transporter with a narrow substrate profile that includes cobalt and zinc. Whereas hepcidin strongly regulated serum iron levels in the mouse, we found no evidence that ferroportin plays an important role in zinc homeostasis.

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 &lt; 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.

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