87 research outputs found
Intracellular pH and K-ATP channel activity in dorsal vagal neurons of juvenile rats in situ during metabolic disturbances
No full textIntracellular pH (pH(i)) is an important factor for understanding cellular processes associated with the response of central neurons to metabolic disturbances such as anoxia or ischemia. In the present study, pH(i) was fluorometrically measured in 2' 7' -bis(carboxyethyl)-5(6)-carboxyfluorescin (BCECF)-filled, voltage-clamped dorsal vagal neurons (DVN) of brainstem slices from rats during metabolic disturbances activating ATP-sensitive K+ (K-ATP) channels. Chemical anoxia induced by cyanide, rotenone or p-trifluoroinethoxy-phenylhydrazone (FCCP) decreased pH(i) by >0.4 pH units. Untreated neurons with normal pH(i) baseline (7.2) responded to glucose-free superfusate after a delay of 7-16 min with a progressive fall of pH(i). In contrast, pHi increased by >0.2 pH units after similar to 10 min in cells that had a mean pH(i) of 6.8 due to incomplete recovery from a CN- induced acid load prior to glucose depletion. Metabolic arrest, induced by cyanide in glucose-free solution after 30 min preincubation in glucose-free saline, caused a progressive glutamate-mediated inward current with no change of pH(i). Upon metabolic arrest, depolarization-evoked pH(i) decreases ( similar to 0.2 pH units) were abolished, whereas glucose-free superfusate slightly delayed their recovery without major effects on amplitude. The glucose-dependent pH(i) fall coincided with activation of the K-ATP channel-mediated outward current, while K-ATP currents due to anoxia or metabolic arrest could reach their maximum in the absence of a major pH(i) change. The results indicate that the anoxic pH(i) decrease is due to enhanced glycolysis and lactate formation with often no obvious effect on K-ATP channel activity. The origin of glucose-dependent acidosis and its relation to K-ATP channel activity remain to be determined. (C) 2004 Elsevier B.V. All rights reserved
ATP-independent anoxic activation of ATP-sensitive K+ channels in dorsal vagal neurons of juvenile mice in situ
No full textThe role of ATP in anoxic activation of ATP-sensitive K+ (K-ATP) channels was studied in dorsal vagal neurons of mouse brainstem slices. In the whole-cell configuration, cyanide-induced chemical anoxia evoked within 10 s a 300-pA outward current that gave rise to a hyperpolarization of 24 mV. These responses were mimicked by nitrogen-aerated saline, rotenone or diazoxide and abolished by tolbutamide. The cyanide-induced hyperpolarization was due to activation of 70 pS K-ATP channels that were half-maximally blocked by 5 muM internal ATP, Dialyzing the cells with either 1, 20 or 0 mM ATP did not, however, affect the time to onset, the kinetics or the magnitude of the cyanide-induced hyperpolarization. Impairment of ATP consumption by ouabain, vanadate or reduced temperature had no effect either. Thus, anoxia-induced activation of these K-ATP channels cannot be explained by a fall of cellular ATP or a concomitant rise of ADP. Anoxia-related changes of the actin cytoskeleton or the composition of the plasma membrane are also not likely to be involved, as cytochalasin D did not affect the cyanide-evoked hyperpolarization and phosphatidylinositol 4,5-bisphosphate failed to decrease the ATP sensitivity of single K-ATP channels. Finally, because of a lack of effects of reduced/oxidized glutathione and the oxidase blocker diphenyliodonium on the cyanide-induced hyperpolarization, cellular redox state does not appear to be involved. Our results indicate that despite a high sensitivity to ATP in excised patches, anoxic activation of K-ATP channels is independent of cellular ATP. Rather the ATP block seems to be removed as a consequence of impaired mitochondrial function. (C) 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved
Anticonvulsant A(1) receptor-mediated adenosine action on neuronal networks in the brainstem-spinal cord of newborn rats
No full textMembrane potential of ventral respiratory group neurons as well as inspiratory-related cranial (hypoglossal) and spinal (C-1-Th-4) nerve activities were analysed in brainstem-spinal cord preparations from neonatal rats. Block of Cl--mediated inhibition with bicuculline (plus strychnine) affected neither rhythmic depolarizations nor spike discharge in 23 of 30 ventral respiratory group cells. In the other seven neurons, block of inhibitory postsynaptic potentials evoked pronounced depolarizations and spike discharge that was synchronous with seizure-like spinal nerve activity. Respiratory hypoglossal nerve activity persisted after transection at the spinomedullary junction, whereas spinal rhythm was blocked. After transection, the moderate bicuculline-evoked seizure-like perturbation of hypoglossal nerve activity was abolished and rhythmic ventral respiratory group neuron activity was not disturbed, whereas epileptiform discharge persisted in spinal nerves. The seizure-like nerve activity and depolarization of the minor subpopulation of perturbed ventral respiratory group neurons were reversed by either adenosine or the A(1) adenosine receptor agonist 2-chloro-N-6-cyclopentyladenosine. The A(2) receptor agonist CGS 21860 had no effect, In control preparations, inspiratory nerve activity and membrane potential fluctuations (29 of 35 cells) were nor changed by adenosine, 2-chloro-N-6-cyclopentyladenosine or CGS 21860. In the other six cells, adenosine evoked a hyperpolarization (<10 mV) with no major change in input resistance. The anticonvulsant effects of adenosine and 2-chloro-N-6-cyclopentyladenosine were antagonized by the A(1) adenosine receptor blocker 8-cyclopentyl-1,3-dipropylxanthine. After pre-incubation with 8-cyclopentyl-1,3-dipropylxanthine, bicuculline also evoked seizure-like discharge in the hypoglossal nerve. The results indicate that seizure-like spinal motor output of the respiratory network upon block of Cl--mediated inhibition is caused by disinhibition of spinal neuronal networks with afferent connections to the ventral respiratory group. Presynaptic Al adenosine receptors exert an anticonvulsant action on the disinhibited spinal motor network, but have no depressing effect per se on the isolated medullary respiratory network. (C) 2000 IBRO. Published by Elsevier Science Ltd
Chemical anoxia activates ATP-sensitive and blocks Ca2+- dependent K+ channels in rat dorsal vagal neurons in situ
No full textThe contribution of subclasses of K+ channels to the response of mammalian neurons to anoxia is not yet clear. We investigated the role of ATP-sensitive (K-ATP) and Ca2+- activated K+ currents (small conductance, SK, big conductance, BK) in mediating the effects of chemical anoxia by cyanide, as determined by electrophysiological analysis and fluorometric Ca2+ measurements in dorsal vagal neurons of rat brainstem slices. The cyanide-evoked persistent outward current was abolished by the KATP channel blocker tolbutamide, but not changed by the SK and BK channel blockers apamin or tetraethylammonium. The K+ channel blockers also revealed that ongoing activation of K-ATP and SK channels counteracts a tonic, spike-related rise in intracellular Ca2+ ([Ca2+](i)) under normoxic conditions, but did not modify the rise of [Ca2+](i) associated with the cyanide-induced outward current. Cyanide depressed the SK channel-mediated afterhyperpolarizing current without changing the depolarization-induced [Ca2+](i) transient, but did not affect spike duration that is determined by BK channels. The afterhyperpolarizing current and the concomitant [Ca2+](i) rise were abolished by Ca2+-free superfusate that changed neither the cyanide-induced outward current nor the associated [Ca2+](i) increase, Intracellular BAPTA for Ca2+ chelation blocked the afterhyperpolarizing current and the accompanying [Ca2+](i) increase, but had no effect on the cyanide-induced outward current although the associated [Ca2+](i) increase was noticeably attenuated. Reproducing the cyanide-evoked [Ca2+](i) transient with the Ca2+ pump blocker cyclopiazonic acid did not evoke an outward current. Our results show that anoxia mediates a persistent hyperpolarization due to activation of KATP channels, blocks SK channels and has no effect on BK channels, and that the anoxic rise of [Ca2+](i) does not interfere with the activity of these K I channels. (C) 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved
An intracellular analysis of gamma-aminobutyric-acid-associated ion movements in rat sympathetic neurones
No full textDouble-barrelled ion-sensitive micro-electrodes were used to measure the changes of the intracellular activities of Cl-, K+, and Na+ (aiCl, aiK, aiNa) in neurones of isolated rat sympathetic ganglia during the action of gamma-aminobutyric acid (GABA). The membrane potential of some of the neurones was manually 'voltage clamped' by passing current through the reference barrel of the ion-sensitive micro-electrode. This enabled us to convert the normal depolarizing action of GABA into a hyperpolarization. A GABA-induced membrane depolarization was accompanied by a decrease of aiCl, aiK and no change in aiNa, whereas a GABA-induced membrane hyperpolarization resulted in an increase of aiCl, aiK and also no change in aiNa. GABA did not change the free intracellular Ca2+ concentration, as measured with a Ca2+-sensitive micro-electrode, whereas such an effect was seen during the action of carbachol. pH-sensitive electrodes, on the other hand, revealed a small GABA-induced extracellular acidification. The inward pumping of Cl- following the normal, depolarizing action of GABA required the presence of extracellular K+ as well as Na+, whereas CO2/HCO3--free solutions did not influence the uptake process. Furosemide, but not DIDS, blocked the inward pumping of Cl-. In conclusion, our data show that only changes in intracellular activities of K+ and Cl- are associated with the action of GABA. Furthermore, they indicate that a K+/Cl- co-transport, and not a Cl-/HCO3- counter-transport, may be involved in the homoeostatic mechanism which operates to restore the normal transmembrane Cl- distribution after the action of GABA
Dynamic recording of cell death in the in vitro dorsal vagal nucleus of rats in response to metabolic arrest
No full textAnoxic/ischemic neuronal death is usually assessed in cell cultures or in vivo within a time window of 24 h to several days using the nucleic acid stain propidium iodide or histological techniques. Accordingly, there is limited information on the time course of such neuronal death. We loaded acute rat brain stem slices with propidium iodide for dynamic fluorometric recording of metabolic arrest-related cell death in the dorsal vagal nucleus. This model was chosen because dorsal vagal neurons show a graded response to metabolic inhibition: anoxia and aglycemia cause a sustained hyperpolarization, whereas ischemia induces a glutamate-mediated, irreversible depolarization. We found that the number of propidium iodide-labeled cells increased from 27% to 43% of total cell count within 1-7 h after preparation of slices. Compared with these untreated control slices, cyanide-induced anoxia (30 min) or aglycemia (1 h) did not cause further cell death, whereas 3-h aglycemia destroyed an additional 13% of cells. Ischemia (1 h) due to cyanide plus iodoacetate immediately labeled an additional 20% of cells, and an additional 48% of cells were destroyed within the following 3 h of postischemia. Continuous recording of propidium iodide fluorescence showed that loss of membrane integrity started within 25 min after onset of the ischemic depolarization and the concomitant intracellular Ca(2+) rise. The results show that propidium iodide can be used to monitor cell death in acute brain slices. Our findings suggest that pronounced cell death occurs within a period of 1-4 h after onset of metabolic arrest and is apparently due to necrotic/oncotic mechanisms
Anoxic persistence of lumbar respiratory bursts and block of lumbar locomotion in newborn rat brainstem-spinal cords
No full textThe tolerance of breathing in neonates to oxygen depletion is reflected by persistence
of inspiratory-related motor output during sustained anoxia in newborn rat brainstem
preparations. It is not known whether lumbar motor networks innervating expiratory abdominal
muscles are, in contrast, inhibited by anoxia similar to locomotor networks in neonatal
mouse lumbar cords. To test this, we recorded inspiratory-related cervical/hypoglossal plus
pre/postinspiratory lumbar/facial nerve activities and, sometimes simultaneously, locomotor
rhythms in newborn rat brainstem–spinal cords. Chemical anoxia slowed 1 : 1-coupled
cervical and lumbar respiratory rhythms and induced cervical burst doublets associated
with depressed preinspiratory and augmented postinspiratory lumbar activities. Similarly,
anoxia evoked repetitive hypoglossal bursts and shifted facial activity toward augmented
postinspiratory bursting in medullas without spinal cord. Selective lumbar anoxia augmented
pre/postinspiratory lumbar bursting without slowing the rhythm. This suggests a medullary
origin of both anoxic inspiratory double bursts and preinspiratory depression, but a mixed
medullary/lumbar origin of boosted postinspiratory lumbar activity. Lumbar respiratory
rhythm is likely to be generated by the parafacial respiratory group expiratory centre as
indicated by lack of normoxic and anoxic bursting following brainstem transection between
the facial motonucleus and the more caudal pre-B¨otzinger complex inspiratory centre. Opposed
to sustained respiratory activities, anoxia reversibly abolished non-rhythmic spinal discharges
and electrically or chemically evoked lumbar locomotor activities, followed by pronounced
postanoxic spinal hyperexcitability. We hypothesize that (i) the anoxia tolerance of neonatal
breathing includes pFRG-driven lumbar expiratory networks, (ii) the anoxic respiratory
pattern transformation is due to disturbed inspiratory–expiratory centre interactions, and (iii)
postanoxic lumbar hyperexcitability contributes to spasticity in cerebral palsy
Changes in intracellular ion activities induced by adrenaline in human and rat skeletal muscle
No full textTo study the stimulating effect of adrenaline (ADR) on active Na+/K+ transport we used double-barrelled ion-sensitive micro-electrodes to measure the activities of extracellular K+ (aKe) and intracellular Na+ (aNai) in isolated preparations of rat soleus muscle, normal human intercostal muscle and one case of hyperkalemic periodic paralysis (h.p.p.). In these preparations bath-application of ADR (10−6 M) resulted in a membrane hyperpolarization and transient decreasesaKe andaNai which could be blocked by ouabain (3×10−4 M). In the h.p.p. muslce a continuous rise ofaNai induced by elevation ofaKe to 5.2 mM could be stopped by ADR. In addition, the intracellular K+ activity (aKi), the free intracellular Ca2+ concentration (pCai) and intracellular pH (pHi) were monitored in rat soleus muscle. During ADRaKi increased, pHi remained constant and intracellular Ca2+ apparently decreased. In conclusion, our data show that ADR primarily stimulates the Na+/K+ pump in mammalian skeletal muscle. This stimulating action is not impaired in the h.p.p. muscle
Role of bicarbonate and chloride in GABA- and glycine-induced depolarization and [Ca2+](i) rise in fetal rat motoneurons in situ
No full textCa2+ imaging and (perforated) patch recording were used to analyze the mechanism of GABA- and glycine-induced depolarizations in lumbar motoneurons of spinal cord slices from fetal rats. In fura-2 ester-loaded cells, the agonist-induced depolarizations increased [Ca2+](i) by up to 100 nM. The GABA- and glycine-evoked [Ca2+](i) transients were suppressed by bicuculline and strychnine, respectively. Their magnitude decreased by similar to 50% between embryonic days 15.5 and 19.5. The [Ca2+](i) increases were abolished by Ca2+-free superfusate and attenuated by similar to 65% by nifedipine, showing that the responses were mediated by voltage-activated Ca2+ channels. The [Ca2+](i) rises were potentiated by >300% immediately after removal of CI- from the superfusate but recovered to values of 50-200% of control during repeated agonist administration in CI--free saline. Bumetanide gradually suppressed the [Ca2+](i) increases by >75%. Subsequent removal of CI- reconstituted the responses and increased, upon repeated agonist application, the peak [Ca2+](i) rises to values above control. Removal of HCO3- from the CI--free (bumetanide-containing) superfusate reversibly abolished both the agonist-induced [Ca2+](i) rises and depolarizations that were reestablished by formate anions. In CI--containing superfusate, removal of HCO3- decreased both the peak and duration of the agonist-evoked membrane depolarization and [Ca2+](i) response. Our findings show that HCO3- efflux has a major contribution to depolarizations mediated by GABAA and glycine receptor-coupled anion channels in prenatal neurons. We hypothesize that the HCO3--dependent depolarizing component, which is likely to produce an intracellular acidosis, might play an important role during the early postnatal period when the CI--dependent component gradually shifts to hyperpolarization
channel function in rat dorsal vagal neurones
No full text1. Using in situ hybridisation histochemistry in combination with patch-clamp, recordings and specific pharmacological tools, the molecular nature of the channels underlying Ca2+- dependent K+ currents was determined in dorsal vagal neurones (DVNs) of rat brainstem slices. 2. In situ hybridisation analysis at cellular resolution revealed the presence of 'big'-conductance Ca2+- and voltage-activated K+ (BK) channel alpha-subunit mRNA, and of only one 'small' conductance Ca2+-activated K+ (SK) channel subunit transcript, SK3, at very high levels in DVNs. By contrast, SK1 and SK2 mRNAs were below the threshold limit of detection. 3. The SK channel-mediated after-hyperpolarizing current (I-AHP) was blocked by apamin with a half-maximal inhibitory concentration of similar to 2.2 nM. This is consistent with homomultimeric SK3 channels mediating I-AHP in DVNs. I-AHP was also blocked by scyllatoxin (20-30 nM) and curare (100-200 mu M). 4. Application of apamin (100 nM) or scyllatoxin (20 nM) invariably caused a substantial increase to 146.1 +/- 10.4 and 181.8 +/- 12.9% of control, respectively in the spontaneous firing rate of DVNs. Action potential duration was not affected by these SK channel blockers. 5. The selective RK channel blocker iberiotoxin (50 nM) increased action potential duration by 22.5 +/- 7.3%, as did low concentrations of tetraethylammonium (0.5 mM; 99.3 +/- 16.4%) and the Ca2+ channel blocker Cd2+ (100 mu M; 49.5 +/- 20.9%). BK. channel blockade did not significantly affect the firing rate of DVNs. 6. These results allow us to establish a tight correlation between the properties of cloned and native BK and SK channels, and to achieve an understanding, at the molecular level, of their role in regulating the spontaneous firing frequency and in shaping single action potentials of central neurones
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