ATP from synaptic terminals and astrocytes regulates NMDA receptors and synaptic plasticity through PSD-95 multi-protein complex.

Recent studies highlighted the importance of astrocyte-secreted molecules, such as ATP, for the slow modulation of synaptic transmission in central neurones. Biophysical mechanisms underlying the impact of gliotransmitters on the strength of individual synapse remain, however, unclear. Here we show that purinergic P2X receptors can bring significant contribution to the signalling in the individual synaptic boutons. ATP released from astrocytes facilitates a recruitment of P2X receptors into excitatory synapses by Ca(2+)-dependent mechanism. P2X receptors, co-localized with NMDA receptors in the excitatory synapses, can be activated by ATP co-released with glutamate from pre-synaptic terminals and by glia-derived ATP. An activation of P2X receptors in turn leads to down-regulation of postsynaptic NMDA receptors via Ca(2+)-dependent de-phosphorylation and interaction with PSD-95 multi-protein complex. Genetic deletion of the PSD-95 or P2X4 receptors obliterated ATP-mediated down-regulation of NMDA receptors. Impairment of purinergic modulation of NMDA receptors in the PSD-95 mutants dramatically decreased the threshold of LTP induction and increased the net magnitude of LTP. Our findings show that synergistic action of glia- and neurone-derived ATP can pre-modulate efficacy of excitatory synapses and thereby can have an important role in the glia-neuron communications and brain meta-plasticity.

P2X receptors and synaptic plasticity.

Adenosine triphosphate (ATP) is released in many synapses in the CNS either together with other neurotransmitters, such as glutamate and GABA, or on its own. Postsynaptic action of ATP is mediated through metabotropic P2Y and ionotropic P2X receptors abundantly expressed in neural cells. Activation of P2X receptors induces fast excitatory postsynaptic currents in synapses located in various brain regions, including medial habenula, hippocampus and cortex. P2X receptors display relatively high Ca2+ permeability and can mediate substantial Ca2+ influx at resting membrane potential. P2X receptors can dynamically interact with other neurotransmitter receptors, including N-methyl-D-aspartate (NMDA) receptors, GABA(A) receptors and nicotinic acetylcholine (ACh) receptors. Activation of P2X receptors has multiple modulatory effects on synaptic plasticity, either inhibiting or facilitating the long-term changes of synaptic strength depending on physiological context. At the same time precise mechanisms of P2X-dependent regulation of synaptic plasticity remain elusive. Further understanding of the role of P2X receptors in regulation of synaptic transmission in the CNS requires dissection of P2X-mediated effects on pre-synaptic terminals, postsynaptic membrane and glial cells.

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.

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 microM) or alpha,beta-methylene ATP (10 microM) 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 (P(Ca)/P(Cs)) 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.

Heterogeneity of the functional expression of P2X3 and P2X2/3 receptors in the primary nociceptive neurons of rat.

The properties and functional expression of the purinergic receptors in small (nociceptive) neurons acutely isolated from the DRG of rat were studied using whole-cell patch-clamp recording. The responses of small DRG neurons to ATP exhibited diverse kinetics and could be subdivided into three types: rapid, slow and mixed kinetics responses. Their affinities to agonists allowed to identify the responsible receptors as P2X3 ("fast") and heteromeric P2X2/3 ("slow") subtypes. The expression of different responses dramatically varied both on the neuron-to-neuron and animal-to-animal basis. Out of 744 neurons tested 24% of cells demonstrated predominance of functional P2X2/3 receptors, 44% had mixed representation and in 32% of cells P2X3 receptors dominated. All the animals tested (110) could be subdivided into 3 groups: in 19% of animals the response of each cell to ATP was mediated by P2X2/3 receptors, both types of ATP-evoked currents were found in 58% of animals and only in 23% of the animals P2X3 receptors dominated. Our results argue with exclusive role of P2X3 receptors in purinergic signaling in primary nociceptive neurons.

Omega-conotoxin GVIA potently inhibits the currents mediated by P2X receptors in rat DRG neurons.

We examined effects of omega-conotoxin previously known as a selective blocker of N-type calcium channels, on the adenosine triphosphate (ATP)-induced currents in the rat dorsal root ganglion neurons. These neurons express at least two types of ionotropic purinoreceptors: P2X3 receptors that have very rapid desensitization kinetics and P2X2/X3 heterooligomeric receptor, which exhibits slow desensitization. We have found that omega-conotoxin GVIA potently inhibits the inward currents mediated by both receptor types. This effect was specific for the receptor subtypes: the IC(50) value for responses evoked by 10 microM ATP was 21.2 +/- 1.7 nM for the P2X3 receptor-mediated responses and 3.84 +/- 0.43 microM for slower responses mediated by P2X2/X3 heteropolymers. The efficacy of another type of omega-conotoxin, MVIIC, is much lower: at 10 microM the latter toxin inhibited the rapidly desensitizing response by 65% and the slowly desensitizing response by 18%. The effects of both toxins were reversible and independent on the membrane potential. Omega-Conotoxin GVIA shifted the dose dependence for the agonistic action of ATP on P2X3 receptors to higher concentrations without producing any effect on the kinetics of the response. It is suggested that omega-conotoxin allosterically modulates the receptor properties, rather than competes for the agonist binding site.

Developmental changes in purinergic calcium signalling in rat neocortical neurones.

The changes in cytoplasm free calcium concentration ([Ca2+]i transients) were measured in Fura-2/AM loaded pyramidal neurones of sensorimotor cortex in acutely prepared slices isolated from 14 days (P14) and 30 (P30) days old rats. Ni2+ (50 microM) diminished the [Ca2+]i transients evoked by 50 mM KCl bath application by 47%+/-8% in neurones of the P14 group and only by 15%+/-6% in those of P30 group (P<0.002). Nifedipine and verapamil in concentration 100 microM reduced the calcium transients amplitude triggered by depolarization to about the same extent in both groups of neurons-on average by 50% and 35%, respectively. The amplitude of [Ca2+]i transients induced by application of 100 microM ATP reached 103+/-6 nM in P14 neurones and 72+/-8 nM in the P30 ones. The ATP-[Ca2+]i induced transient could be evoked in Ca2+-free external solution, indicating the presence of metabotropic (P2y) purinoreceptors. Almost all (90%) P14 neurones were endowed with such receptors. At the same time only 1/3 of the tested (n=42) P30 neurones presented responses to ATP applications in Ca2+-free solution. The share of ionotropic (P2x) purinoreceptors in generation of calcium signal was the same in both groups of neurons. No caffeine-induced Ca2+-release has been observed in the P14 neurons. To the contrary, in 28 cells from 42 investigated neurones of the P30 group application of 40 mM caffeine for 10 s induced considerable [Ca2+]i transients, which did not disappear in calcium-free solution. A conclusion is made about substantial changes in the expression of Ca2+-handling mechanisms which are occurring in neocortical neurones at the third-fourth week of postnatal development.

Iono- and metabotropically induced purinergic calcium signalling in rat neocortical neurons.

ATP receptor-mediated Ca2+ concentration changes were recorded from neocortical neurones in brain slices from 2 week-old rats. To measure the cytoplasmic concentration of Ca2+ ([Ca2+]i) slices were incubated with fura-2/AM, and the microfluorimetry system was focused on an individual cell. During transients the intracellular level of [Ca2+]i in the majority of neocortical neurones (98 of 102) varied in the concentration range of ATP 5-2000 microM between 41. 3+/-5 and 163+/-7 nM. The rank order of efficacy for purinoreceptor agonists in concentration 100 microM was: ATPgammaS>ATP>ADP>>AMP approximately Adenosine approximately alpha,beta-methylene ATP>UTP. 10 microM PPADS, a P2-purinoreceptor antagonist, reduced the ATP-induced [Ca2+]i response by 26%+/-4%. After elimination of calcium from extracellular solution the first ATP-induced [Ca2+]i transient decreased to 65+/-8%, suggesting the participation of metabotropic P2y triggered Ca-release in the generation of the transient. Elevation of cytosolic Ca2+ by activation of plasmalemmal Ca2+ channels failed to potentiate such release indicating the absence of effective reloading of the corresponding stores. No Ca2+-induced Ca2+-release has been observed in the investigated neurons.

Steady magnetic fields effect on lipid peroxidation kinetics.

The effect of steady magnetic fields (ranging from 0 to 280 mT) has been investigated on the kinetics of non-enzymatic lipid peroxidation occurring in a model system consisting of liposomes obtained from 1, 2-dioleoylphosphatidylcholine by oxygen consumption. The process was found to be accelerated by weak steady magnetic fields. A computer simulation method was employed to detect the reactions that govern the process kinetics, to elucidate magneto-sensitive stages (initiation and reduction of iron(III), as well as lipid peroxide radical recombination) and to determine their rate constants at various external magnetic fields. The kinetics of peroxidation of lipid cell membranes have been modeled mathematically at oxygen and 'free' iron concentrations close to those in the cells and also at increased free iron concentrations at different external magnetic field values.