Yuriy Pankratov

Quantal release of ATP in mouse cortex

Transient currents occur at rest in cortical neurones that reflect the quantal release of transmitters such as glutamate and γ-aminobutyric acid (GABA). We found a bimodal amplitude distribution for spontaneously occurring inward currents recorded from mouse pyramidal neurones in situ, in acutely isolated brain slices superfused with picrotoxin. Larger events were blocked by glutamate receptor (AMPA, kainate) antagonists; smaller events were partially inhibited by P2X receptor antagonists suramin and PPADS. The decay of the larger events was selectively prolonged by cyclothiazide. Stimulation of single intracortical axons elicited quantal glutamate-mediated currents and also quantal currents with amplitudes corresponding to the smaller spontaneous inward currents. It is likely that the lower amplitude spontaneous events reflect packaged ATP release. This occurs with a lower probability than that of glutamate, and evokes unitary currents about half the amplitude of those mediated through AMPA receptors. Furthermore, the packets of ATP appear to be released from vesicle in a subset of glutamate-containing terminals.

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.

Exocytosis of ATP From Astrocytes Modulates Phasic and Tonic Inhibition in the Neocortex

Astrocytes secrete ATP by exocytosis from synaptic-like vesicles, activating neuronal P2X receptors, which contribute to postsynaptic GABA receptor down-regulation, ultimately mediating the communication between astrocytes and neurons required for brain function.

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.

Ionotropic NMDA and P2X1/5 receptors mediate synaptically induced Ca2+ signalling in cortical astrocytes

Local, global and propagating calcium (Ca(2+)) signals provide the substrate for glial excitability. Here we analyse Ca(2+) permeability of NMDA and P2X(1/5) receptors expressed in cortical astrocytes and provide evidence that activation of these receptors trigger astroglial Ca(2+) signals when stimulated by either endogenous agonists or by synaptic release of neurotransmitters. The Ca(2+) permeability of the ionotropic receptors was determined by reversal potential shift analysis; the permeability ratio P(Ca)/P(K) was 3.1 for NMDA receptors and 2.2 for P2X(1/5) receptors. Selective stimulation of ionotropic receptors (with NMDA and α,β-methyleneATP) in freshly isolated cortical astrocytes induced ion currents associated with transient increases in cytosolic Ca(2+) concentration ([Ca(2+)](i)). Stimulation of neuronal afferents in cortical slices triggered glial synaptic currents and [Ca(2+)](i) responses, which were partially blocked by selective antagonists of NMDA (D-AP5 and UBP141) and P2X(1/5) (NF449) receptors. We conclude that ionotropic receptors contribute to astroglial Ca(2+) signalling and may provide a specific mechanism for fast neuronal-glial signalling at the synaptic level.

miR-132/212 knockout mice reveal roles for these miRNAs in regulating cortical synaptic transmission and plasticity

miR-132 and miR-212 are two closely related miRNAs encoded in the same intron of a small non-coding gene, which have been suggested to play roles in both immune and neuronal function. We describe here the generation and initial characterisation of a miR-132/212 double knockout mouse. These mice were viable and fertile with no overt adverse phenotype. Analysis of innate immune responses, including TLR-induced cytokine production and IFNβ induction in response to viral infection of primary fibroblasts did not reveal any phenotype in the knockouts. In contrast, the loss of miR-132 and miR-212, while not overtly affecting neuronal morphology, did affect synaptic function. In both hippocampal and neocortical slices miR-132/212 knockout reduced basal synaptic transmission, without affecting paired-pulse facilitation. Hippocampal long-term potentiation (LTP) induced by tetanic stimulation was not affected by miR-132/212 deletion, whilst theta burst LTP was enhanced. In contrast, neocortical theta burst-induced LTP was inhibited by loss of miR-132/212. Together these results indicate that miR-132 and/or miR-212 play a significant role in synaptic function, possibly by regulating the number of postsynaptic AMPA receptors under basal conditions and during activity-dependent synaptic plasticity.

Ionotropic receptors in neuronal–astroglial signalling: What is the role of “excitable” molecules in non-excitable cells

Astroglial cells were long considered to serve merely as the structural and metabolic supporting cast and scenery against which the shining neurones perform their illustrious duties. Relatively recent evidence, however, indicates that astrocytes are intimately involved in many of the brains functions. Astrocytes possess a diverse assortment of ionotropic transmitter receptors, which enable these glial cells to respond to many of the same signals that act on neurones. Ionotropic receptors mediate neurone-driven signals to astroglial cells in various brain areas including neocortex, hippocampus and cerebellum. Activation of ionotropic receptors trigger rapid signalling events in astroglia; these events, represented by local Ca(2+) or Na(+) signals provide the mechanism for fast neuronal-glial signalling at the synaptic level. Since astrocytes can detect chemical transmitters that are released from neurones and can release their own extracellular signals, gliotransmitters, they are intricately involved in homocellular and heterocellular signalling mechanisms in the nervous system. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Distinct pharmacological and functional properties of NMDA receptors in mouse cortical astrocytes

BACKGROUND AND PURPOSE Astrocytes of the mouse neocortex express functional NMDA receptors, which are not blocked by Mg2+ ions. However, the pharmacological profile of glial NMDA receptors and their subunit composition is far from complete.

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.

MSK1 Regulates Homeostatic and Experience-Dependent Synaptic Plasticity

The ability of neurons to modulate synaptic strength underpins synaptic plasticity, learning and memory, and adaptation to sensory experience. Despite the importance of synaptic adaptation in directing, reinforcing, and revising the behavioral response to environmental influences, the cellular and molecular mechanisms underlying synaptic adaptation are far from clear. Brain-derived neurotrophic factor (BDNF) is a prime initiator of structural and functional synaptic adaptation. However, the signaling cascade activated by BDNF to initiate these adaptive changes has not been elucidated. We have previously shown that BDNF activates mitogen- and stress-activated kinase 1 (MSK1), which regulates gene transcription via the phosphorylation of both CREB and histone H3. Using mice with a kinase-dead knock-in mutation of MSK1, we now show that MSK1 is necessary for the upregulation of synaptic strength in response to environmental enrichment in vivo. Furthermore, neurons from MSK1 kinase-dead mice failed to show scaling of synaptic transmission in response to activity deprivation in vitro, a deficit that could be rescued by reintroduction of wild-type MSK1. We also show that MSK1 forms part of a BDNF- and MAPK-dependent signaling cascade required for homeostatic synaptic scaling, which likely resides in the ability of MSK1 to regulate cell surface GluA1 expression via the induction of Arc/Arg3.1. These results demonstrate that MSK1 is an integral part of a signaling pathway that underlies the adaptive response to synaptic and environmental experience. MSK1 may thus act as a key homeostat in the activity- and experience-dependent regulation of synaptic strength.