Enhancement of glutamate release uncovers spillover-mediated transmission by N-methyl-D-aspartate receptors in the rat hippocampus.

Properties of excitatory postsynaptic currents during increased glutamate release were investigated by means of a whole-cell voltage-clamp in CA1 pyramidal neurons of rat hippocampal slices. Enhancement of transmitter release by 50 microM 4-aminopyridine or by elevated extracellular Ca2+ (up to 5 mM) resulted in a substantial increase in the peak excitatory postsynaptic current amplitude and in the significant stimulus-dependent prolongation of the excitatory postsynaptic current decay. The stronger the stimulus, the slower the excitatory postsynaptic current decay became. The pharmacologically isolated N-methyl-D-aspartate, but not alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid component of the excitatory postsynaptic current exhibited this phenomenon. The possible connection of such behaviour of the N-methyl-D-aspartate component to the loss of voltage control was tested in the following way: the peak of the N-methyl-D-aspartate component was enhanced under 50 microM 4-aminopyridine and then returned back to the control level by a low dose of D-2-amino-5-phosphonopentanoic acid. However, the decay of the decreased N-methyl-D-aspartate component remained slow suggesting another origin of the stimulus-dependent kinetics. Dihydrokainate, a non-competitive inhibitor of glutamate uptake, did not influence the kinetics of the N-methyl-D-aspartate component in control but induced its dramatic stimulus-dependent prolongation when applied on the background of a low dose of 4-aminopyridine (10 microM) which did not affect the decay by itself. We propose that the delayed stimulus-dependent kinetics of the N-methyl-D-aspartate component is due to the saturation of uptake mechanisms and subsequent activation of extrasynaptic N-methyl-D-aspartate receptors. Our present observations therefore support the hypothesis that N-methyl-D-aspartate receptors may play a role in the cross-talk between synapses by means of the transmitter spillover.

Phorbol diacetate differentially regulates the N-methyl-D-aspartate (NMDA) and non-NMDA receptor-mediated components of the rat hippocampal excitatory postsynaptic currents.

The effects of phorbol 12,13-diacetate (PDAc) on evoked excitatory transmission were studied in neurons of the CA1 area of hippocampal slices of rats, using whole-cell voltage clamp of pyramidal neurons in situ and stimulation of the Schaffer collaterals. The application of PDAc (10 microM) increased the amplitude of the excitatory postsynaptic current (EPSC) and caused a lengthening of its decay, due to an increase in the contribution of the N-methyl-D-aspartate (NMDA) component to the total EPSC. The latter effect was depend upon the concentration of calcium in the extracellular medium. Experiments in which we separated the two components of the EPSCs by 6-cyano-7-nitroquinoxaline-2,3-dione and by 2-amino-5-phosphonopentanoic acid also demonstrated a more pronounced increase in the NMDA receptor-mediated current under PDAc. The effects of PDAc were markedly attenuated by the extracellular application of the protein kinase C inhibitor H-7 (300 microM), but not by intracellular perfusion with 20 mM of the same drug.

Intracellular ATP modulates desensitization of acetylcholine receptors controlling chloride current in Lymnaea neurons.

Chloride current activated by nicotinic acetylcholine receptors (AChR) was examined in dialysed voltage-clamp neurons of Lymnaea stagnalis. Fast superfusion of acetylcholine (ACh) evoked an inward current rapidly rising to a peak followed by a decline due to desensitization. When adenosine triphosphate with Mg2+ (MgATP, 2-10 mM) was added intracellularly the peak of the ACh-induced current was increased and its decay was slowed down. ATP without Mg2+ did not affect desensitization. Mg2+ alone accelerated desensitization. Intracellular treatment with an inhibitor of ATP synthesis, sodium arsenate, increased the desensitization rate and decreased the peak current. MgATP after arsenate wash-out restored the initial characteristics of the response; a mixture of glycolytic substrates had a similar effect. A non-hydrolysable analogue of ATP, adenosine [gamma-thio]triphosphate mimicked ATP action after arsenate removal but was weaker; another non-hydrolysable analogue, adenylyl imidodiphosphate, did not affect desensitization at all. Intracellular treatment of the neurons with alkaline phosphatase accelerated current decay. The data suggest that a change in intracellular ATP concentration modulates AChR desensitization via an enzymatic process that might be phosphorylation of AChR or some associated protein(s). Involvement of Ca2+ homeostasis cannot be excluded. The results are compared with the data obtained on vertebrate tissues under conditions promoting phosphorylation.

Intracellular ATP modifies the voltage dependence of the fast transient outward K+ current in Lymnaea stagnalis neurones.

1. The action of intracellular ATP on the fast transient outward K+ current (A-current) was studied in dialysed voltage-clamped Lymnaea stagnalis neurones. 2. When introduced intracellularly in millimolar concentrations ATP caused a shift of the steady-state inactivation curve along the voltage axis in the direction of positive potentials and decreased A-current at all test voltages. 3. Intracellular treatment with an inhibitor of ATP synthesis, sodium arsenate, led to the opposite changes. The action of arsenate was not reversed upon its removal. After wash-out of arsenate ATP restored the initial voltage dependence. 4. Addition of Mg2+ to the solution weakened the action of ATP in proportion to the Mg2+: ATP concentration ratio. On the other hand, in neurones pretreated with arsenate, Mg2+ did not affect the ATP action. 5. When a mixture of glycolytic substrates was applied after arsenate wash-out the activation and inactivation curves shifted towards positive voltages. A substrate of oxidative phosphorylation was ineffective in the same conditions. 6. Non-hydrolysable analogues of ATP, adenosine-5'-O-gamma-thiotriphosphate and adenylyl imidodiphosphate, did not mimic the ATP action. This means that the ATP effect is mediated by some enzymatic process(es). 7. Elevation of total cytosolic Ca2+ concentration as well as intracellular application of agents increasing intracellular free Ca2+ reduced A-current amplitude but failed to alter its voltage dependence. Therefore, ATP action cannot be related to activation of Ca2+ transport. 8. Treatment of the neurones with alkaline phosphatase evoked a shift of the inactivation voltage dependence towards hyperpolarizing potentials and increased the A-current amplitudes at all test voltages. 9. The data indicate that a change in intracellular ATP concentration modulates the A-current voltage dependence. The effect of ATP is probably the result of phosphorylation of a channel protein or some associated proteins, but lowering of free Mg2+ concentration cannot be excluded. The possible physiological significance of the phenomenon is discussed.

Intracellular Mg2+ modulates the A-current and its blockage by catechol in isolated Lymnaea neurons.

The effects of intracellular Mg2+ (2-8 mM) upon the transient outward current (the A-current) under normal conditions and under catechol-induced blockage were studied in molluscan neurons by using the voltage-clamp and intracellular dialysis techniques. Identified giant Lymnaea stagnalis L. neurons were investigated at room temperature (20-22 degrees C). When applied intracellularly, Mg2+ caused both time- and dose-dependent shifts of the voltage dependence of the steady-state activation and inactivation of the A-current to more negative membrane potentials. Upon external application, catechol suppressed (5-6 mM) or eliminated (9-10 mM) the A-currents, slowed down the current decay and shifted the activation and inactivation curves to more positive membrane voltages. Intracellular Mg2+ decreased the blocking ability of extracellularly applied catechol, whereas catechol antagonized the Mg2(+)-induced negative shift of the steady-state activation and inactivation curves of the A-currents.