Manipulation of synaptic sign and strength with divalent cations in the vertebrate retina: pushing the limits of tonic, chemical neurotransmission

At the first synaptic level of the vertebrate retina, photoreceptor light responses are transmitted to second order neurones through a chemical synapse based on a tonic release of neurotransmitter modulated by graded changes of presynaptic potential. The possibility that such synapses could work through a Ca2+-independent process had been proposed by previous authors, based on the persistence of transmission process in low Ca2+ media containing Co2+ or Ni2+ ions. Recently, we were able to explain these results within the framework of the classical calcium-hypothesis of synaptic transmission by taking into account the modifications of presynaptic surface potential brought about by changes of divalent cation concentrations. Here we report data showing how a surface-charge hypothesis could account for several apparently paradoxical effects of divalent cation manipulations such as: the enhancement of neurotransmitter release induced by low Ca2+ media; the transmission "unblocking" effect of Zn2+, Co2+ and Ni2+; and the reversal of transmission polarity induced by application of low Ca2+ media containing Cd2+ or Mg2+ ions.     

Inhibition of cardiac inward-rectifier K current by terodiline

The antispasmodic agent terodiline has cardiotoxic effects that include QT lengthening. To determine whether inhibition of inwardly-rectifying K⁺ current (I_K1) might be a factor in the cardiotoxicity, we measured I_K1 in guinea pig ventricular myocytes. Terodiline reduced outward I_K1 with an IC₅₀ of 7 μM; maximal reduction was 60% with 100–300 μM concentration. Inhibition was independent of current direction, and persisted after removal of the drug. Terodiline (3–5 μM) lengthened action potentials in guinea pig papillary muscles by ca. 10%, primarily by slowing phase 3 repolarization; higher concentrations abbreviated the plateau and markedly slowed late repolarization. Terodiline washout provoked an extra lengthening, consistent with persistent inhibition of I_K1 and rapid recovery of net inward plateau current. The results suggest that inhibition of I_K1 is a likely factor in the cardiotoxicity of the drug.     

Inhibition of potassium outward currents and pacemaker current in sheep cardiac Purkinje fibres by the verapamil derivative YS 035

Summary The electrophysiologic mode of action and potency of the verapamil derivative YS 035 (N,N-bis-(3,4-dimethoxyphenethyl)-N-methyl amine) were investigated in sheep cardiac Purkinje fibres. Action potential duration measured at a repolarization level of –60 mV (APD-60) and membrane currents recorded with the two-microelectrode voltage-clamp technique were evaluated. At 10 mol/l YS 035 APD-60 was increased to about 115% of reference. Prolongation measured as percentage of the respective control exhibited on the average no dependence on stimulation frequency (0.17–2 Hz). At 100 mol/l membrane became depolarized to about –50 mV and action potentials could no longer be elicited. Further study was focussed on effects on outward currents, mostly activated at a frequency of 0.05 Hz. Transient outward current (i_to) was completely blocked at 100 mol/l and half-maximal inhibition occurred at about 14 mol/l. Inwardly rectifying potassium current (i_K1) was reduced to 47% of reference at 100 mol/l. An initially activating outward current at positive membrane potentials (i_inst) was reduced to 73% at 100 mol/l. Time-dependent (delayed) outward current (i_K) was on the average not affected up to 100 ol/l. Besides inhibition of repolarizing outward currents YS 035 completely blocked pacemaker current (i_f) at 100 mol/l and half-maximal reduction was achieved at 5 mol/l. YS 035 (1–100 mol/l) did not clearly affect time constants of activation at selected test potentials (I_K: +35 mV; i_f: –90 mV) or inactivation (i_to: 0 mV). Voltage-dependent control mechanisms of currents (it_to, i_f) were not influenced by YS 035 but the amount of available current was reduced.In conclusion, the verapamil derivative YS 035 inhibited pacemaker current and potassium outward currents which correlated to a prolongation of cardiac action po tentials. Electrophysiological actions of the compound favour it to be tested in vivo as an antiarrhythmic drug candidate. Send offprint requests to U. Borchard at the above address     

Further characterization of the effects of imipramine on plateau membrane currents in guinea-pig ventricular myocytes

Summary The effects of imipramine, a tricyclic antidepressant, on action potential characteristics and plateau membrane currents were studied in isolated guineapig ventricular myocytes, using the whole cell configuration of the patch-clamp technique. Imipramine (1, 5 and 15 mol/l) decreased in a concentration-dependent manner the amplitude and shortened the duration of the action potential, but it had no effect on resting membrane potential. At all three concentrations tested, imipramine decreased the delayed outward potassium current, this effect being apparently voltage-independent since it did not modify the activation curve. Imipramine, 5 and 15 mol/l, also produced an inhibition of the peak high threshold calcium current, but did not change the shape of the current-voltage relationship or the apparent reversal potential of this current. Therefore, imipramine probably decreased the maximum available calcium conductance. However, the inward rectifying potassium current was not affected by any concentration of imipramine tested. Imipramine, 1 and 5 mol/l, shortened the duration of the action potentials elicited in the presence of the inorganic calcium channel blocker cobalt chloride, and at 5, but not at 1 mol/l, also shortened the action potentials obtained in the presence of the sodium channel blocker tetrodotoxin. Washout of imipramine completely reversed all its effects within 15 minutes. All these results suggest that imipramine at a concentration of 1 mol/l produced a shortening in action potential duration by inhibiting the late sodium current flowing during the plateau phase of the action potential. At concentrations of 5 and 15 mol/l the effect of imipramine on action potential duration can also be explained by a blocking effect on the high threshold calcium current. Send offprint requests to E. Delpón at the above address     

Effects of metoprolol on action potential and membrane currents in guinea-pig ventricular myocytes

Summary The effects of the beta-adrenoceptor antagonist metoprolol on action potentials and membrane currents were studied in single guinea-pig ventricular myocytes. The experiments were carried out using the nystatin-method of whole-cell technique. This method was used in order to prevent the run-down of the calcium current. Metoprolol at concentrations of 10–100 mol/l shortened action potential in a dose-dependent way. The drug only decreased resting membrane potential at a concentration of 100 mol/1 in two out of five cells. Under voltage-clamp conditions, metoprolol blocked the high threshold calcium current at concentrations of 30 and 100 mol/l to 82 ± 4% and 73 ± 5% from control, respectively. The drug decreased the inward rectifying potassium current in a concentration-dependent manner. This effect was evident for inward current at voltages negative to the apparent reversal potential and for outward current at voltages between –30 and –80 mV. This blocking effect on the inward rectifying potassium current can explain the effect on resting membrane potential. At voltages positive to –30 mV metoprolol increased a time-independent outward current. This metoprolol-enhanced outward current was blocked by barium and cesium. This result suggests that the metoprolol-enhanced current is carried by potassium. The current component enhanced by metoprolol was not sensitive to glibenclamide and tetraethylammonium applied externally, which suggests that the adenosine triphosphate-sensitive channel is not the target of metoprolol. The activation of this time-independent outward current by metoprolol and the blocking effects on the calcium current seem to explain the shortening in action potential induced by the drug. Send offprint requests to J. Sánchez-Chapula at the above address     

GABA-mediated synaptic transmission in neuroendocrine cells: a patch-clamp study in a pituitary slice preparation

Patch-clamp recording techniques were applied to thin slices of the rat pituitary gland in order to study synaptic transmission between hypothalamic nerve terminals and neuroendocrine cells of the intermediate lobe. Inhibitory postsynaptic currents (IPSCs) could be evoked by electrical stimulation of afferent neuronal fibres in the surrounding tissue of the slice. The IPSCs could be evoked in an all-or-nothing mode depending on the stimulus intensity, suggesting that single afferent fibres were stimulated. They had a chloride-dependent reversal potential and were blocked by bicuculline (K _d=0.1 M), indicating that they were mediated by -aminobutyric acid A (GABA_A) receptors. In symmetrical chloride solutions the current/voltage relation of the IPSC peak amplitudes was linear. The IPSCs were characterized by a fast (1–2 ms) rise time and a biexponential decay, with time constants of 21±4 ms and 58±14 ms at a holding potential of –60 mV (n=6 cells). Both decay time constants increased with depolarization in an exponential manner. Spontaneously occurring IPSCs had a time course that was similar to that of evoked IPSCs. These miniature IPSCs, recorded in 1 M tetrodotoxin, displayed an amplitude distribution that was well fitted by single Gaussian functions, with a mean value of its maxima of 18.1±2.3 pA (n=4 cells). Amplitude histograms of evoked IPSCs were characterized by multiple peaks with a modal amplitude of about 18 pA (n=6 cells). These findings indicate the quantal nature of GABAergic synaptic transmission in this system, with a quantal conductance step of about 280 pS. Single-channel currents underlying the IPSCs were studied by bath application of GABA to outside-out patches excised from intermediate lobe cells. Such GABA-induced currents revealed two conductance levels of 14 pS and 26 pS. In conclusion, GABAergic synaptic transmission in neuroendocrine cells of the pituitary has properties that are quite similar to those observed in neurones of the central nervous system.     

The effect of arachidonic acid on the M current of NG108-15 neuroblastoma × glioma hybrid cells

The M current, I _M, a voltage-dependent non-inactivating K⁺ current, was recorded in NG108-15 neuroblastoma × glioma hybrid cells, using the whole-cell mode of the patch-clamp technique. We studied the effect of arachidonic acid, other fatty acids and inhibitors of the arachidonic acid metabolism. In relatively high concentrations (25–50 M) arachidonic acid first increased and later decreased the current, I _h, which holds the membrane potential at –30 mV and mainly flows through open M channels. It shifted the midpoint potential, V _o, of the relation between M conductance, g _M, and membrane potential, V, to more negative values and decreased the maximum conductance ¯g _M and the time constant _M. In smaller concentrations (5–10 M) arachidonic acid merely decreased I _h and ¯g _M with little effect on V _o and _M. Eicosatetraynoic acid and docosa-hexaenoic acid acted similarly to arachidonic acid whereas stearic acid had no effect. Of the three enzyme inhibitors studied, nordihydroguaiaretic acid acted similarly to arachidonic acid. i. e. caused a biphasic change in I _h. Indomethacin and quinacrine caused, respectively, a pure increase and a pure decrease of I _h and ¯g _M. Possible explanations are build-up of internally produced arachidonic acid, depletion of eicosanoid products or an inhibitory effect unrelated to arachidonic acid metabolism.     

A loudspeaker-driven system for rapid and multiple solution exchanges in patch-clamp experiments

A new and inexpensive system allowing rapid and synchronized changes of solutions around a membrane patch or a cell under voltage-clamp conditions is described. Four plastic capillary tubings (OD 640 m; ID 430 m) were glued together horizontally and attached to a coil of a commercially available loudspeaker. Servo-control of the position of the coil allowed the mouth of any of the capillaries to be positioned near the pipette tip within 6 ms. A high flow speed of the test solution was crucial to achieve rapid solution exchange. At a flow speed of 5 cm/s, complete exchange of the external environment of a frog ventricular cell was achieved within 20–30 ms. The time course of solution change was found to be 3–5 times faster at the tip of an open patch pipette. To preserve the physical integrity of the cell, the cell was usually perfused by a control capillary at a slow velocity (0.2 –0.4 cm/s) and test solutions flowing out of adjacent capillaries at high velocity (4–5 cm/s) were applied to the cell only for short periods. Determination of the three-dimensional contamination profile around the mouth of the control capillary allowed the optimal conditions for the use of the system to be established and possible sources of contamination to be avoided between adjacent capillaries with unmatched flow speeds. Successive and multiple changes in external solutions could be easily synchronized with voltage-clamp depolarizations to examine the time course of the effect of drugs on voltage-operated ion channels. An example of this application is given with rapid applications of the dihydropyridine agonist (-)BayK 8644 to the L-type Ca²⁺ channel current in frog ventricular myocytes.     

"Conflict" situation based on intracranial self-stimulation behavior and the effect of benzodiazepines

Based on lateral hypothalamic self-stimulation behavior of the rat in a Skinner box, a "conflict" situation was established by combining foot shock punishment with brain stimulation. Diazepam (10-20 mg/kg, PO) caused a marked increase in the lever pressing response in the punished period without affecting the unpunished response. Bromazepam (10--20 mg/kg PO) also caused an increase in the lever pressing response in the punished period and a decrease of the punished response. These results indicate that a "conflict" situation based on self-stimulation behavior is useful for the evaluation of antianxiety action.     

The relation between the current underlying pacemaker activity and beta-adrenoceptors in cardiac Purkinje fibres: A study using adrenaline,procaine, atenolol and penbutolol

Summary The pacemaker current — i _K2 — in cardiac Purkinje fibres was analysed using the voltage clamp technique described by Deck et al. (1964). (–)-Adrenaline (5.5 · 10^–6 M) causes the wellknown shift of the Hodkin-Huxley kinetics in the depolarizing direction. Procaine (7.3·10^–4 M) does not cause any further shift of s in the presence of adrenaline. Atenolol (3.8·10^–5 M) causes a backshift of the kinetics in the negative direction in the presence of adrenaline and procaine. The instantaneous current-voltage relationship ( ) is altered neither with adrenaline, nor with procaine or atenolol. The results exclude the possibility that the local anaesthetic side effect of many beta-adrenoceptor blocking agents may be involved in the backshift of the s-kinetics. The voltage dependence of the reciprocals of the time constants is shifted in a similar way as s by the sympathomimetic or blocking drugs. Following the application of (–)-adrenaline (5.5·10^–6 M) the (–)-isomere of penbutolol (1.7 and 3.5·10^–6 M) is about equally effective in shifting the kinetics back as the (+)-isomere (3.5·10^–5 M). In the presence of (–)-adrenaline, the (+)- and (–)-forms of penubutolol cause virtually no change of the instantaneous current-voltage relationship, . Thus, (–)-adrenaline and (+)- and (–)-penbutolol are aiming for the s-kinetics whose voltage dependence is controlled by the electric field near the i _K2-channel of the membrane and do not influence the number of the i _K2-channels. These findings suggest that the sympathomimetic or blocking agents influence the s-kinetics of the pacemaker current i _K2 by altering the electric field; the fully activated current-voltage relationship which is proportional to the number of the open i _K2-channels is not subject to any appreciable modification. The results conclusively show that the kinetics of the pacemaker current can be controlled by beta-adrenoceptors.