mV In Cells Research Articles

A quantitative analysis of glial cell coupling in the retina of the axolotl ( Ambystoma mexicanum)

The strength of gap junctional coupling of radial glial cells (Mu¨ller cells) in the isolated axolotl retina was assessed by monitoring the spread of dye between cells, and by injecting current into one cell and recording the voltage response in surrounding cells. Dye injected into one Mu¨ller cell spread to surrounding Mu¨ller cells, and could be detected up to 130 μm away, i.e. over 4 times the mean Mu¨ller cell spacing of 30 μm. Injecting 1 nA of current into a Mu¨ller cell evoked responses of 7 mV in that cell, 1 mV in next neighbour cells, and 0.2 mV in cells at 60 μm distance. Analysis of these data indicates an electrical space constant for the Mu¨ller cell network of 15 μm, and predicts that isolated cells should have a resistance of 11.4 MΩ. Mu¨ller cells isolated by papain dissociation of the retina were found, by whole-cell patch-clamping, to have a mean resistance of 12.4 MΩ. These results on lateral coupling are combined with data showing that over 90% of the Mu¨ller cell potassium conductance is in the vitreal endfoot of these cells to provide a fairly complete electrical description of the radial glial cell network in the retina. Gap junctional coupling of Mu¨ller cells increases by 60% the ‘spatial buffering’ that these glial cells can carry out to reduce localized rises in extracellular potassium concentration. The location of the majority of the Mu¨ller cell potassium conductance in the cell endfoot ensures that laterally buffered K + is deposited in the vitreous, rather than depolarizing surrounding retinal neurones.

Neurotransmitter‐induced currents in retinal bipolar cells of the axolotl, Ambystoma mexicanum.

1. Whole-cell patch clamping was used to study the membrane properties of isolated bipolar cells and the currents evoked in them by putative retinal neurotransmitters. 2. Isolated bipolar cells show an approximately ohmic response to voltage steps over most of the physiological response range, with an average input resistance of 1.3 G omega and resting potential of -35 mV. These values are underestimates because of the shunting effect of the seal between the patch electrode and the cell membrane. Depolarization beyond -30 mV produces rapid activation (10-100 ms) of an outward current (carried largely by potassium ions), which then inactivates slowly (0.5-2 s). 3. Of five candidates for the photoreceptor transmitter, four (aspartate, N-acetylhistidine, cadaverine, putrescine) had no effect on bipolar cells. The fifth substance, L-glutamate, opened ionic channels with a mean reversal potential of -12 mV in some cells (presumed hyperpolarizing bipolar cells), and closed channels with a mean reversal potential of -13 mV in other cells (presumed depolarizing bipolar cells). 4. The conductance increase induced by glutamate in presumed hyperpolarizing bipolar cells was associated with an increase in membrane current noise. Noise analysis suggested a single-channel conductance for the glutamate-gated channel of 5.4 pS. The power spectrum of the noise increase required the sum of two Lorentzian curves to fit it, suggesting that the channel can exist in three states. 5. The conductance decrease induced by glutamate in presumed depolarizing bipolar cells was associated with a decrease in membrane current noise that could be described as the sum of two Lorentzian spectra, and which suggested a single-channel conductance of 11 pS. The noise decrease implies that the channels closed by glutamate are not all open in the absence of the transmitter. 6. GABA (gamma-aminobutyric acid) and glycine, transmitters believed to mediate lateral inhibition in the retina, open chloride channels in isolated bipolar cells, and increase the membrane current noise. Noise analysis suggested that the channels gated by GABA and glycine have conductances of 4.4 and 7.5 pS respectively. The noise spectra required the sum of two Lorentzian curves to fit them. 7. By whole-cell patch clamping cells in retinal slices, the synaptic transmitter released by photoreceptors was shown to close channels with an extrapolated reversal potential around -3 mV in depolarizing bipolar cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Two Types of Action Potentials in A Secretory Epithelium of A Clam Mantle

ABSTRACT The secretory epithelium that faces the shell of the clam Anomalocardia brasiliana is electrically excitable. It was found that two types of action potentials could be elicited by electrical stimulation. In mantles showing signs of being in the phase of shell secretion, cardiac-like action potentials were observed. They had an overshoot of 48·4 ±8·8mV (±S.D.), a rate of rise of 54·7 ±5·6 Vs−1 and three phases of repolarization of 9·3 ± 4·8, 1·44 ± 0·70 and 4·7 ± 1·1 Vs−1. Their duration averaged 38·4 ± 13·3 ms. Mantles of clams resorbing the shell had spike-like action potentials with an overshoot of 38·7 ± 5·5 mV, a rate of rise of 52·7 ± 16·8 Vs−1, and a single phase of repolarization of 12·4 ± 2·5 Vs−1. Their duration was 11·1 ± l’4ms. It is suggested that the presence of a plateau may be related to shell secretion. Significant differences were also found in the resting potentials: −54·8±5·4mV in cells of mantles with cardiac-like responses and −62·5 ±4·9 mV in cells of mantles with spikes. In both types of mantles the action potentials were propagated throughout the epithelium and both exhibited a refractory period. Lowered Ca or increased frequency of stimulation reduced the plateau of cardiac-like action potentials without any marked alteration in the resting potential, rate of rise, or overshoot. The action potential was elicited by outward (depolarizing) current across the basolateral membrane, and not by similarly-directed current across the apical membrane, so we conclude that the basolateral membrane is the excitable one.

Two components of muscarine-sensitive membrane current in rat sympathetic neurones.

Membrane currents induced by muscarine (Imus) were recorded in voltage-clamped neurones in isolated rat superior cervical ganglia. Two components of Imus were regularly recorded: an inward current resulting from inhibition of the outward K+ current, IM; and an outward current attributable to the reduction of a steady inward current. The presence of these two components caused a 'cross-over' in the current-voltage curves at -50 +/- 3 mV in neurones impaled with KCl-filled micro-electrodes or at -63 +/- 4 mV in neurones impaled with K-acetate-filled electrodes. Both components of Imus were prevented by atropine. Both persisted in Krebs solution containing tetrodotoxin (1 microM), Cd2+ (200 microM) or 0 Ca2+. When IM was inhibited by external Ba2+ or internal Cs+ only the outward component of Imus could be detected. This component reversed at +3 +/- 2 mV in cells impaled with CsCl-filled electrodes or at -20 +/- 3 mV in cells impaled with Cs-acetate-filled electrodes. The reversal potentials agreed with those for the currents induced by gamma-aminobutyric acid (+4 +/- 2 mV and -16 +/- 3 mV with CsCl and Cs acetate electrodes respectively). Replacement of external NaCl with Na acetate (so reducing external Cl- concentration ( [Cl-]o) from 155 to 22 mM) shifted the reversal potential for Imus by +25 and +14.5 mV in two cells impaled with CsCl-filled electrodes. A tenfold reduction of external [Na+] (by glucosamine replacement) did not significantly alter the reversal potential for Imus in KCl or CsCl-impaled cells. Under conditions where IM is already inhibited, the residual outward component of Imus can lead to hyperpolarization and inhibition of neuronal activity in unclamped cells. We conclude that both inward and outward components of Imus result from direct activation of muscarinic receptors on the ganglion cells. The inward component results from IM inhibition. We suggest that the outward component results from inhibition of another, voltage-independent current IX which largely comprises a Cl- current. The inward component induces membrane depolarization and an increased excitability; the outward component can lead to hyperpolarization and reduced excitability.

Estimation of the cytoplasmic pH of Coxiella burnetii and effect of substrate oxidation on proton motive force

The magnitude of the proton motive force generated during in vitro substrate oxidation by Coxiella burnetii was examined. The intracellular pH of C. burnetii varied from about 5.1 to 6.95 in resting cells over an extracellular pH range of 2 to 7. Similarly, delta psi varied from about 15 mV to -58 mV over approximately the same range of extracellular pH. Both components of the proton motive force increased during substrate oxidation, resulting in an increase in proton motive force from about -92 mV in resting cells to -153 mV in cells metabolizing glutamate at pH 4.2. The respiration-dependent increase in proton motive force was blocked by respiratory inhibitors, but the delta pH was not abolished even by the addition of proton ionophores such as carbonyl cyanide-m-chlorophenyl hydrazone or 2,4-dinitrophenol. Because of this apparently passive component of delta pH maintenance, the largest proton motive force was obtained at an extracellular pH too low to permit respiration. C. burnetii appears, therefore, to behave in many respects like other acidophilic bacteria. Such responses are proposed to contribute to the extreme resistance of C. burnetii to environmental conditions and subsequent activation upon entry into the phagolysosome of eucaryotic cells in which this organism multiplies.

Physiological response properties of cells labeled intracellularly with horseradish peroxidase in cat dorsal cochlear nucleus.

The physiology and morphology of fusiform cells in the dorsal cochlear nucleus were studied using extracellular and intracellular recording and intracellular injection of horseradish peroxidase. Fusiform cells displayed a variety of responses to tone pips presented at the characteristic frequency; most often these cells exhibited the pauser/buildup pattern defined in earlier studies. The response pattern of each neuron was dependent on frequency and sound-pressure level. Tone pips evoked short-lasting depolarizations of about 10 mV and long-lasting hyperpolarizations of about 10 mV in cells whose resting potentials were -50 to -65 mV. The time courses of both the excitation and the inhibition depended on frequency and sound-pressure level. Generally the depolarization was sustained for the duration of the tone pip, whereas the hyperpolarization could last as long as 600 ms after the end of the tone pip. Often a neuron exhibited a sustained chopper pattern after microelectrode impalement. This was probably a result of a decrease in membrane potential which altered the relative effectiveness of the excitatory and inhibitory inputs. The large, bitufted fusiform cells had many apical dendrites, which branched one to five times and were covered with spines, and fewer basal dendrites, which exhibited little branching and had few appendages. The morphology of fusiform cells varied systematically as a function of location within the dorsal cochlear nucleus. Response patterns for tone pips were not exclusive to individual cell types as two nonfusiform cells were found to exhibit a buildup pattern. Axons of injected neurons left the nucleus via the dorsal acoustic stria and 14 of 15 had collaterals within the dorsal cochlear nucleus.

Effects of aerobiosis and nitrogen source on the proton motive force in growing Escherichia coli and Klebsiella pneumoniae cells.

The electrochemical gradient of hydrogen ions, or proton motive force (PMF), was measured in growing Escherichia coli and Klebsiella pneumoniae in batch culture. The electrical component of the PMF (delta psi) and the chemical component (delta pH) were calculated from the cellular accumulation of radiolabeled tetraphenylphosphonium, thiocyanate, and benzoate ions. In both species, the PMF was constant during exponential phase and decreased as the cells entered stationary phase. Altering the growth rate with different energy substrates had no effect on the PMF. The delta pH (alkaline inside) varied with the pH of the culture medium, resulting in a constant internal pH. During aerobic growth in media at pH 6 to 7, the delta psi was constant at 160 mV (negative inside). The PMF, therefore, was 255 mV in cells growing at pH 6.3, and decreased progressively to 210 mV in pH 7.1 cultures. K. pneumoniae cells and two E. coli strains (K-12 and ML), including a mutant deficient in the H+-translocating ATPase and a pleiotropically energy-uncoupled mutant with a normal ATPase, had the same PMF during aerobic exponential phase. During anaerobic growth, however, both species had delta psi values equal to 0. Therefore, the PMF in anaerobic cells consisted only of the delta pH component, which was 75 mV or less in cells growing at pH 6.2 or greater. These data thus met the expectation that cells deriving metabolic energy from respiration have a PMF above a threshold value of about 200 mV when the ATPase functions in the direction of H+ influx and ATP synthesis; in fermenting cells, a PMF below a threshold value was expected since the enzyme functions in the direction of H+ extrusion and ATP hydrolysis. K. pneumoniae cells growing anaerobically had no delta psi whether the N source added was N2, NH+4 or one of several amino acids; the delta pH was unaffected. Therefore, any energy cost incurred by the process of nitrogen fixation could not be detected as an alteration of the proton gradient.

Proton motive force in growing Streptococcus lactis and Staphylococcus aureus cells under aerobic and anaerobic conditions.

Measurements of the electrochemical gradient of hydrogen ions, which gives rise to the proton motive force (PMF), were carried out with growing Streptococcus lactis and Staphylococcus aureus cells. The facultative anaerobe was chosen in order to compare the PMF of cells growing aerobically and anaerobically. It was expected that during aerobic growth the cells would have a higher PMF than during anaerobic growth, because the H+-translocating ATPase (BF0F1) operates in the direction of H+ influx and ATP synthesis during respiration, whereas under anaerobic conditions the BF0F1 hydrolyzes glycolytically generated ATP and establishes the proton gradient by extruding H+. The electrical component of the PMF, delta psi, and the chemical gradient of H+, delta pH, were measured with radiolabeled tetraphenylphosphonium and benzoate ions. In both S. lactis and S. aureus cells, the PMF was constant during the exponential phase of batch growth and decreased in the stationary phase. In both species of bacteria, the exponential-phase PMF was not affected by varying the growth rate by adding different sugars to the medium. The relative contributions of delta psi and delta pH to the PMF, however, depended on the pH of the medium. The internal pH of S. aureus was constant at pH 7.4 to 7.6 under all conditions of growth tested. Under aerobic conditions, the delta psi of exponential phase S. aureus remained fairly constant at 160 to 170 mV. Thus, the PMF was 250 to 270 mV in cells growing aerobically in media at pH 6 and progressively lower in media of higher pH, reaching 195 to 205 mV at pH 7. Under anaerobic conditions, the delta psi ranged from 100 to 120 mV in cells at pH 6.3 to 7, resulting in a PMF of 150 to 140 mV. Thus, the mode of energy metabolism (i.e., respiration versus fermentation) and the pH of the medium are the two important factors influencing the PMF of these gram-positive cells during growth.

Actions of noradrenaline and acetylcholine on sympathetic ganglion cells.

1. The responses of the post-synaptic membrane of sympathetic ganglion cells to noradrenaline (NA) and to acetylcholine (ACh) were studied in relation to the slow inhibitory post-synaptic potential (S-IPSP) and slow excitatory one (S-EPSP) respectively.2. NA produced an hyperpolarization of about 4 mV in cells of rabbit superior cervical ganglia.3. The hyperpolarizing response to NA was not accompanied by any detectable change in membrane resistance, and it was depressed by conditioning depolarization.4. NA also depressed all the post-synaptic potentials, presumably by an action on presynaptic function.5. ACh produced a large depolarization in ganglion cells of rabbit and of frog (paravertebral) ganglia, which was accompanied by a large decrease in membrane resistance.6. When ACh was applied during nicotinic blockade, achieved with high concentration of nicotine (frog ganglia) or D-tubocurarine (rabbit ganglia), it still produced a considerable depolarization. This response could be blocked by atropine, and is presumably a muscarinic type of action.7. The muscarinic-ACh response was not accompanied by a decrease in membrane resistance. Instead, the frog ganglion cells exhibited increased resistances of up to more than twice the resting value during both the muscarinic-ACh depolarization and the S-EPSP.8. The muscarinic-ACh depolarization and the S-EPSP were both depressed by conditioning hyperpolarization (in nicotinized frog cells). An initial hyperpolarizing phase now appeared in both of these responses.9. It is concluded that the hyperpolarizing response to NA and the depolarizing response to muscarinic-ACh action are not generated by increases in ionic mobilities in the post-synaptic membrane; and that these two responses are produced by the same electrogenic mechanisms which underlie the S-IPSP and the S-EPSP respectively.