Oleg Krishtal

Excitatory amino acid receptors in hippocampal neurons: Kainate fails to desensitize them

The excitatory responses to L-glutamate (L-Glu), quisqualate (QA) and kainate (KA) have been investigated in isolated pyramidal cells from rat hippocampus using intracellular perfusion and concentration clamp techniques. The responses to L-Glu and QA demonstrated rapid desensitization and complete cross-desensitization, while KA produced a non-desensitizing response. The activation of KA response was determined by the level of desensitization induced by L-Glu or QA pretreatment. It is concluded that QA, KA and L-Glu activate the same excitatory receptors with apparent Kd values of 9.3 X 10(-5) M, 5.0 X 10(-4) M and 1.1 X 10(-3) M, respectively.

Ionotropic P2X purinoreceptors mediate synaptic transmission in rat pyramidal neurones of layer II/III of somato‐sensory cortex

Fast P2X receptor‐mediated excitatory postsynaptic current (EPSC) was identified in pyramidal neurones of layer II/III of somato‐sensory cortex in acutely isolated slices obtained from the brain of 17‐ to 22‐day‐old rats. The EPSCs were elicited by electrical stimulation of vertical axons originating from layer IV‐VI neurones at 0.1 Hz in the presence of bicuculline. When the glutamatergic EPSC was blocked by saturating concentrations of glutamate receptor inhibitors 2,3‐dioxo‐6‐nitro‐1,2,3,4‐tetrahydrobenzo‐[f]‐quinoxaline‐7‐sulphonamide (NBQX) and D‐(‐)‐2‐amino‐5‐phosphonopentanoic acid (D‐AP5), a small EPSC component was recorded from 90 % of neurones tested. This residual EPSC was not affected by selective blockers of nicotinic (hexamethonium) or serotonin (N‐(1‐azabicyclo‐[2.2.2]oct‐3‐yl)‐6‐chloro‐4‐methyl‐3‐oxo‐3,4‐dihydro‐2H‐1,4‐benzoxazine‐8‐carboxamide hydrochloride, Y‐25130) receptors, but it was reversibly inhibited by the antagonists of P2X receptors NF023 (8,8′‐[carbonylbis(imino‐3,1‐phenylenecarbonylimino)]bis‐1,3,5‐naphthalene‐trisulphonic acid), NF279 (8,8′‐[carbonylbis (imino‐4,1‐phenylenecarbonylimino‐4,1‐phenylenecarbonylimino)]bis‐1,3,5‐naphthalene‐trisulphonic acid) and PPADS (pyridoxal phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid). Application of ATP (10 μm) or α,β‐methylene ATP (10 μm) to pyramidal neurones, acutely isolated from cortical slices, evoked inward currents (30 to 200 pA) in 65 % of cells tested. The relative calcium/caesium permeability (PCa/PCs) of P2X receptors was 12.3 as estimated from the reversal potential of ATP‐induced current measured at different extracellular calcium concentrations. We concluded that P2X purinoreceptors are activated during synaptic transmission in neocortex.

Comparative Patch‐clamp Studies with Freshly Dissociated Rat Hippocampal and Striatal Neurons on the NMDA Receptor Antagonistic Effects of Amantadine and Memantine

Patch‐ and concentration‐clamp techniques were used to compare the effects of the uncompetitive N‐methyl‐D‐aspartate (NMDA) receptor antagonists (+)‐MK‐801 (dizocilpine, (+)‐5‐methyl‐10, 11‐dihydro‐5H‐dibenzo‐cyclohepten‐5, 10‐imine maleate), ketamine, memantine (1‐amino‐3, 5‐dimethyladamantane) and amantadine (1‐amino‐adamantane) on agonist‐induced inward currents in freshly dissociated rat hippocampal and striatal neurons. In hippocampal neurons, ketamine (5 μM), memantine (10 μM) and amantadine (100 μM) selectively antagonized inward current responses to NMDA (500 μM plus glycine 5 μM) in a voltage‐dependent manner without affecting responses to (s)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazoleproprionic acid (100 μM) or γ‐aminobutyric acid (10 μM). The NMDA receptor antagonistic effect of all four agents was typical of open channel blockade. The kinetics of blockade/unblockade was inversely related to antagonist affinity. In hippocampal neurons amantadine was the least potent NMDA receptor antagonist (IC50 18.6 ± 0.9 μM) and showed the fastest blocking kinetics, whereas (+)‐MK‐801 was the most potent (IC50 0.12 ± 0.01 μM) and showed the slowest blocking kinetics. Memantine (IC50 1.04 ± 0.26 μM) and ketamine (IC50 0.43 ± 0.10 μM) were almost equipotent and had similar, intermediate blocking kinetics. In striatal neurons recorded under identical conditions (+)‐MK‐801, ketamine and memantine were 3‐ to 4‐fold less potent whereas amantadine was somewhat more potent than on hippocampal neurons. This could offer an explanation for the better clinical profile of amantadine in Parkinsons disease, as therapeutically relevant concentrations of amantadine are likely to be more active in the striatum whereas memantine is likely to be more active in other structures.

P2X receptor-mediated excitatory synaptic currents in somatosensory cortex

Fast P2X receptor-mediated excitatory postsynaptic current (EPSC) was found in pyramidal neurones of layer V of somatosensory cortex in slices acutely isolated from the brain of 17- to 22-day-old rats. The EPSCs were elicited by field electrical stimulation in the layer VI at 0.1 Hz in the presence of picrotoxin. When the glutamatergic EPSC was blocked by glutamate receptors inhibitors NBQX and D-AP5, a residual EPSC (rEPSC) was recorded from 85% of neurones tested. This rEPSC was not affected by blockers of nicotinic (hexamethonium) and serotonin (Y25130) receptors; however, it was reversibly inhibited by P2X receptors antagonists (NF023, NF279, and PPADS). An application of ATP (20 microM), beta,gamma-methylene ATP (25 microM), and alpha,beta-methylene ATP (20 microM) to acutely isolated pyramidal neurones of layer V evoked inward currents (30 to 400 pA) in 75% of cells tested. We concluded that several subtypes of P2X purinoreceptors participate in synaptic transmission in neocortex.

Cross-desensitization Reveals Pharmacological Specificity of Excitatory Amino Acid Receptors in Isolated Hippocampal Neurons.

Ionic currents elicited by excitatory amino acids were studied, using the concentration clamp method, in enzymatically isolated rat hippocampal neurons. Cross‐desensitization between the responses to various agonists was applied to separate the activity of two types of receptors, N‐methyl‐D‐aspartate (NMDA) and non‐NMDA. NMDA receptors were selectively activated by NMDA, L‐ and D‐aspartate, D‐glutamate and quinolinate. Kainate and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate appeared to be selective, and quisqualate relatively less selective non‐NMDA agonists, acting on the same receptor type. L‐Glutamate, L‐ and D‐homocysteate activated both receptor types. It is supposed that two receptor sites, activation site and densensitization site, control the action of agonists at the non‐NMDA receptor. When examined in the cross‐desensitization experiments, NMDA and non‐NMDA receptors appear to be represented by the two homogeneous and independent receptor populations operating different ionic channels.

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.

Possible functional role of diadenosine polyphosphates: Negative feedback for excitation in hippocampus

Diadenosine polyphosphates (Ap4A and Ap5A) are present in secretory granules of chromaffin cells as well as in the rat brain synaptic terminals. Their contribution to the exocytosis of the total synaptosomal content is considerable, ranging from 7% to 12%. Ap4A and Ap5A are released from synaptosomes in a Ca(2+)-dependent manner. There are indications on the high affinity of diadenosine polyphosphates to P2 receptors, but their action on P1 receptors remains unclear. Here we report that both substances induce a blocking action on excitatory synaptic transmission in the rat hippocampus. This action is elicited via the A1 (subclass of P1) receptors and differs in some respects from the action of adenosine.

Chapter 19 ATP receptor-mediated component of the excitatory synaptic transmission in the hippocampus

Publisher Summary This chapter presents the pharmacological identification of a purinergic component in the excitatory synaptic input to CA1 neurons of the rat hippocampus. The investigation is initiated by the observation that the specific P2-purinoceptor antagonist pyridoxal phosphate-6-azophenyI-2’-4’-disulfonic acid (PPADS) had a small, but consistent and reversible, inhibitory effect on the excitatory post-synaptic currents (EPSC) recorded in CA1 pyramidal neurons in response to stimulation of Schaffer collateral at especially low stimulation frequencies (0.1–0.05 Hz). The current paradigm establishes that the excitatory synaptic transmission in hippocampal CAl/CA3 synapses is mediated solely by excitatory amino acid receptors. The chapter demonstrates the presence of a clear non-glutamatergic component in the EPSC measured in CA1 pyramidal neurons. Based on the selectivity of PPADS and the sensitivity to Zn2 + and ATP analogs the chapter concludes that the transmitter underlying this component is most likely ATP acting on ionotropic P2X receptors. The chapter reveals that about one fifth of the excitatory input to CA1 neurons is purinergic.

Distinct Quantal Features of AMPA and NMDA Synaptic Currents in Hippocampal Neurons: Implication of Glutamate Spillover and Receptor Saturation

Excitatory postsynaptic currents (EPSCs) were studied in the CA1 pyramidal cells of rat hippocampal slices. Components mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) and by N-methyl-D-aspartate (NMDA) receptors were separated pharmacologically. Quantal parameters of AMPA and NMDA receptor-mediated EPSCs were obtained using both maximal likelihood and autocorrelation techniques. Enhancement of transmitter release with 4-aminopyridine caused a significant increase in quantal size of NMDA EPSC. This was accompanied by a slowing of the EPSC decay. The maximal number of quanta in the NMDA current was unchanged, while the probability of quantal event dramatically enhanced. In contrast, neither the quantal size nor the kinetics of AMPA EPSC was altered by 4-aminopyridine, while the maximal number of quanta increased. These changes in the quantal parameters are consistent with a transition to multivesicular release of the neurotransmitter. Spillover of excessive glutamate on the nonsynaptic areas of dendritic spines causes an increase in the quantal size of NMDA synaptic current. The difference in quantal behavior of AMPA and NMDA EPSCs implies that different mechanisms underlie their quantization: the additive response of nonsaturated AMPA receptors contrasts with the variable involvement of saturated intrasynaptic and nonsaturated extrasynaptic NMDA receptors.

BN52021, a platelet activating factor antagonist, is a selective blocker of glycine-gated chloride channel

We have found that the platelet activating factor antagonist (BN52021) is an effective blocker of the glycine (Gly) receptor-mediated responses in the hippocampal pyramidal neurons of rat. Using the whole-cell voltage clamp and concentration clamp recording techniques, we investigated the mechanism underlying the inhibitory action of this terpenoid on the glycine-induced chloride current. BN52021 selectively and reversibly inhibits glycine current in a non-competitive and voltage-dependent fashion. The antagonistic effect of this substance is more pronounced at positive membrane potentials. At holding potential -70mV and in the presence of 200 microM glycine IC50 value for the blocking action of BN52021 was 270+/-10nM. Repetitive applications of BN52021 reveal the use-dependence of its blocking action. When co-applied with strychnine (STR), a competitive glycine receptor antagonist, BN52021 does not alter the IC50 value for strychnine. The inhibitory effect of BN52021 on gamma-aminobutyric acid (GABA) current is at least 25 times less potent than the effect on glycine current. This substance fails to affect AMPA and NMDA responses. It may be concluded that BN52021 inhibits glycine-gated Cl- channels by interacting with the pore region and does not compete for the strychnine-binding centre.