Day care surgery for prominent ears.
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Adolescent↗
Biomedical subjects
A R Kay
Publications and source records attributed to A R Kay.
Errors in persistent inward currents generated by space-clamp errors: a modeling study.
1. The effects of imperfect space clamp on inactivating inward currents were examined with the use of a "ball-and-stick" neuronal model with uniform active and passive membrane properties. With poor space clamp, both transient and steady-state (persistent) components were distorted. The ratio of steady-state to peak current (i(s)/p), measured at the soma, was sometimes smaller but usually larger than would be the case with uniform space clamp. For a fast Na+ current, the anomalous persistent component was largest for large electrotonic lengths, low-conductance densities, and voltage-clamp potentials near the threshold of the current. Under some conditions, steady-state current could take one of two values, depending on the holding potential. 2. Membrane potential as a function of distance was examined, revealing a steady-state voltage gradient in which distal portions of the neuron were more positive than in the passive case, and often more positive than the command potential itself. These reversed voltage gradients, caused by the uncontrolled "window" Na+ current at remote electrotonic distances, produced steady-state axial current flow into the soma, thereby increasing the persistent current measured somatically. 3. The time at which the current peaked (tp) was sensitive to imperfections in the space clamp. This phenomenon made somatic membrane current and axial current at tp sensitive to the fidelity of space clamp as well. The ratio of steady-state axial current to that at t = tp was a good predictor of the degree of distortion of i(s)/p.(ABSTRACT TRUNCATED AT 250 WORDS)
Kinetics↗
Hyperpolarization-activated Na(+)-K+ current (Ih) in neocortical neurons is blocked by external proteolysis and internal TEA.
1. After 1 day in culture, neurons derived from the neonatal cerebral cortex of the rat exhibited a slowly activating current gated by hyperpolarizing voltage clamp pulses. The current was blocked by extracellular Cs+ and unaffected by extracellular Ba2+, and was permeable to both Na+ and K+ (PNa/PK = 0.29). Its form and pharmacology are consistent with a current termed Ih in other preparations. 2. Ih was absent from cells acutely dissociated from both the neonatal and mature cerebral cortex, despite the use of low enzyme concentrations. The sensitivity of Ih to extracellular proteolysis was demonstrated by superfusing the cells with trypsin (1 mg/ml) while monitoring the presence of Ih in the whole cell mode of recording. Ih was rapidly abolished (t1/2 approximately 5 min at 22 degrees C) by proteolysis and exhibited no shifts in its range of activation or changes in its activation kinetics during the course of the digestion. 3. Intracellular tetraethylammonium (TEA), at a concentration of < 15 mM was shown to block Ih completely, while extracellular TEA had no effect on the current. This suggests that the inner vestibule of Ih may be structurally related to that of potassium channels.
Animals↗
An intracellular medium formulary.
Whole-cell patch-clamp recordings allow diffusible intracellular ions and molecules to be replaced by the contents of the recording pipette. In this review, the formulation of intracellular media is considered with a view to improving the stability of recordings and emulating the intracellular environment.
Animals↗
Microsurgical toenail transfer to the hand.
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Fingers↗
Inactivation kinetics of calcium current of acutely dissociated CA1 pyramidal cells of the mature guinea-pig hippocampus.
1. The process of inactivation of the Ca2+ current of acutely dissociated pyramidal cells from the CA1 subfield of mature guinea-pig hippocampus was characterized. The decline of the current after rapid activation could be approximated well by the sum of two exponentials (time constants approximately 200 ms and 2 s) and a constant offset. 2. The time constants of inactivation exhibited a voltage dependence consistent with a voltage-dependent mechanism. However, under conditions which normally counteract Ca(2+)-dependent inactivation (viz. intracellular bis(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) and external Ba2+) all three showed a U-shaped inactivation curve, characteristic of Ca(2+)-dependent inactivation. 3. The rate of inactivation was found to increase with current at a given voltage; however, increasing external divalent ion concentrations did not accelerate inactivation. 4. Calcium imaging experiments, using the Ca(2+)-sensitive probe, Fura-2, were performed to estimate the accumulation of Ca2+ in the presence of 10 mM-intracellular BAPTA. Under these conditions voltage steps which induced maximal Ca2+ currents lead to free Ca2+ concentrations of less than 500 nM in the bulk of the cytoplasm. 5. Elevation of the intracellular free Ca2+ concentration to above 1 microM suppressed all the components of the Ca2+ current. However, even at a concentration of 3 microM-Ca2+ the U-shaped inactivation curve persisted. 6. Substitution of Ca2+ for Ba2+ led to an acceleration of inactivation through an increase in the proportion of the fast process of inactivation and an acceleration of both the fast and slow rates of inactivation. 7. During the slow decline of Ca2+ current ('run-down') the proportion of all three components remained approximately constant and there was little change in the rate of inactivation. 8. On the basis of the results I suggest that inactivation results fro a dual process of voltage- and Ca(2+)-dependent inactivation. Ca(2+)-dependent inactivation seems to result from the accumulation of Ca2+ close to the channel mouth. 9. The macroscopic properties of the Ca2+ channel are consistent with the existence of one channel type in the CA1 pyramidal cells.
Animals↗
Preservation of the NMDA response of neurons acutely dissociated from the mature guinea pig hippocampus.
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Animals↗
Brain magnetic resonance imaging with contrast dependent on blood oxygenation.
Paramagnetic deoxyhemoglobin in venous blood is a naturally occurring contrast agent for magnetic resonance imaging (MRI). By accentuating the effects of this agent through the use of gradient-echo techniques in high fields, we demonstrate in vivo images of brain microvasculature with image contrast reflecting the blood oxygen level. This blood oxygenation level-dependent (BOLD) contrast follows blood oxygen changes induced by anesthetics, by insulin-induced hypoglycemia, and by inhaled gas mixtures that alter metabolic demand or blood flow. The results suggest that BOLD contrast can be used to provide in vivo real-time maps of blood oxygenation in the brain under normal physiological conditions. BOLD contrast adds an additional feature to magnetic resonance imaging and complements other techniques that are attempting to provide positron emission tomography-like measurements related to regional neural activity.
Animals↗
GABAA receptor function is regulated by phosphorylation in acutely dissociated guinea-pig hippocampal neurones.
1. Current mediated by GABAA receptors was examined in pyramidal cells acutely dissociated from the hippocampus of mature guinea-pigs. Current responses were measured using whole-cell voltage-clamp recordings. An internal perfusion technique was used to change the intracellular contents during recording. 2. Application of GABA (100-300 microM) by short duration pressure pulses produced outward current responses at a holding potential of -10 mV. When recordings were made with intracellular solutions which did not contain Mg-ATP, GABA responses progressively decreased to less than 10% of their initial values after 10 min. This 'run-down' of the GABA response could not be accounted for by desensitization since the rate of run-down was not dependent upon agonist application. 3. The run-down of the GABAA response was reversed when Mg2+ (4 mM) and ATP (2 mM) were introduced into the intracellular perfusate. In addition to the presence of Mg-ATP, buffering of Ca2+ in the intracellular solution to low levels (approximately 10(-8) M) was also necessary to stabilize the GABAA response. 4. The role of a phosphorylation process in regulating the GABAA receptor was tested. After the GABA response stabilized, introduction of alkaline phosphatase (100 micrograms/ml) to the intracellular perfusate caused a complete run-down of the GABA response. 5. Stable GABA responses were obtained when ATP was replaced by ATP-gamma-S (adenosine 5'-O-(thiotriphosphate), an analogue of ATP that donates a thiophosphate group resulting in a product that is more resistant to hydrolysis. Following such treatment GABA responses declined more slowly after the introduction of intracellular alkaline phosphatase. 6. Run-down of GABA responses accelerated when intracellular Ca2+ concentration ([Ca2+]i) was elevated to about 5 x 10(-4) M. The run-down caused by elevated [Ca2+]i could be stopped and reversed by reducing [Ca2+]i to about 10(-8) M. 7. The introduction of ATP-gamma-S to the intracellular medium retarded the run-down of GABA responses caused by elevation of [Ca2+]i. 8. N-(6-Aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7), a calmodulin inhibitor, reduced the rate of run-down induced by elevated [Ca2+]i. 9. These results suggest that the function of the GABAA receptor is maintained by phosphorylation of the receptor or some closely associated regulatory molecule. Elevation of [Ca2+]i destabilizes the function of the GABAA receptor, probably by activating a Ca2+/calmodulin-dependent phosphatase.
Adenosine Triphosphate↗
GABAA-receptor function in hippocampal cells is maintained by phosphorylation factors.
Gamma aminobutyric acid (GABA) mediates fast synaptic inhibition in the central nervous system by activating the chloride-permeable GABAA channel. The GABAA conductance progressively diminishes with time when the intracellular contents of hippocampal neurons are perfused with a minimal intracellular medium. This "run down" of the GABA-activated conductance can be prevented by the inclusion of magnesium adenosine triphosphate and calcium buffer in the intracellular medium. The amount of chloride conductance that can be activated by GABA is determined by competition between a calcium-dependent process that reduces the conductance and a phosphorylation process that maintains the conductance.
Adenosine Triphosphate↗
Calcium current activation kinetics in isolated pyramidal neurones of the Ca1 region of the mature guinea-pig hippocampus.
1. Neurones were isolated from the CA1 region of the guinea-pig hippocampus and subjected to the whole-cell mode of voltage clamping, to determine the kinetics of voltage-gated Ca2+ channel activation. 2. Isolated neurones had an abbreviated morphology, having lost most of the distal dendritic tree during the isolation procedure. The electrical compactness of the cells facilitates voltage clamp analysis. 3. Block of sodium and potassium currents revealed a persistent current activated on depolarization above -40 mV, which inactivated slowly when the intracellular medium contained EGTA. The current was blocked by Co2+ and Cd2+, augmented by increases in Ca2+ and could be carried by Ba2+, suggesting that the current is borne by Ca2+. 4. Steady-state activation of the Ca2+ current was found to be well described by the Boltzman equation raised to the second power. 5. The open channel's current-voltage (I-V) relationship rectified in the inward direction and was consistent with the constant-field equation. 6. The kinetics of Ca2+ current onset followed m2 kinetics throughout the range of its activation. Tail current kinetics were in accord with this model. A detailed Hodgkin-Huxley model was derived, defining the activation of this current. 7. The kinetics of the currents observed in this regionally and morphologically defined class of neurones were consistent with the existence of a single kinetic class of channels.
Action Potentials↗
Isolation of neurons suitable for patch-clamping from adult mammalian central nervous systems.
A method is described for the isolation of neurons from defined regions of the mammalian central nervous system, by a combination of mechanical and enzymatic means. The procedure liberates neurons free of cellular debris and glial investments, allowing the formation of giga-ohm seals with patch clamp electrodes. The characteristic morphology of neurons is maintained, together with the diversity of active channels evident in the intact nervous system.
Aging↗
Intracellular fluoride alters the kinetic properties of calcium currents facilitating the investigation of synaptic events in hippocampal neurons.
We have attempted to suppress voltage-dependent conductances in hippocampal neurons by introducing various intracellular agents. Voltage-clamp studies were carried out using acutely dissociated hippocampal neurons from adult guinea pigs. Synaptic events were examined using intracellular recordings in the slice preparation. Sodium conductance was suppressed when the quaternary lidocaine derivative QX 314 was introduced intracellularly. Potassium conductances were blocked by intracellular cesium or Tris. We also found that the anion fluoride could affect calcium conductance by an intracellular action. When anions other than fluoride were used for intracellular recordings, the voltage-dependent calcium current inactivated slowly and showed persistent activation at membrane potentials between -40 and -10 mV. In contrast, when fluoride was present intracellularly, the inactivation kinetics of the calcium current were accelerated and the persistent component of the current was largely suppressed. Intracellular recordings in the hippocampal slice showed that when electrodes contained cesium, QX 314, and fluoride, the spiking and nonlinear responses of the neuronal membrane to depolarization were blocked. In these conditions the time course and voltage-dependence of EPSPs could be examined in detail without complications due to voltage-dependent currents of the postsynaptic cell.
Animals↗
Single cyclic GMP-activated channel activity in excised patches of rod outer segment membrane.
The plasma membrane of retinal rod outer segments contains a cyclic GMP-activated conductance which appears to be the light-sensitive conductance involved in phototransduction. Recently, it has been found that this conductance is partially blocked by Mg2+ and Ca2+ at physiological concentrations, thus possibly accounting for the absence of observable single-channel activity in excised membrane patches and for the unusually small apparent unit conductance deduced from noise measurements on intact cells. We now report that, as expected from this idea, single cGMP-activated channel activity can be detected from an excised rod membrane patch in the absence of divalent cations. The most prominent unitary current had a mean conductance of approximately 25 pS. Both individual channel openings (mean open time approximately 1 ms) and short bursts of openings (mean burst duration of about a few milliseconds) were observed. In addition, there were smaller events which probably represented other states of the conductance. The mean current increased with the third power of cGMP concentration, suggesting that there are at least three cGMP-binding sites on the channel molecule. With 0.2 mM Mg2+ in the cGMP-containing solution, a flickering block of the open channel was observed; the effect of Ca2+ was similar. The results resolve a puzzle about the light-sensitive conductance by demonstrating that it is an aqueous pore rather than a carrier.
Ambystoma↗