P2X7-like receptor subunits enhance excitatory synaptic transmission at central synapses by presynaptic mechanisms

doi:10.1016/j.neuroscience.2004.06.014

Neuroscience

Volume 128, Issue 2, 2004, Pages 269-280

https://doi.org/10.1016/j.neuroscience.2004.06.014 Get rights and content

Abstract

Recent studies demonstrate that P2X₇ receptor subunits (P2X₇RS) are present at central and peripheral synapses and suggest that P2X₇RS can regulate transmitter release. In brainstem slices from 15 to 26 day old pentobarbitone-anesthetized mice, we examined the effect of P2X₇RS activation on excitatory postsynaptic currents (EPSCs) recorded from hypoglossal motoneurons using whole-cell patch clamp techniques. After blockade of most P2X receptors with suramin (which is inactive at P2X₇RS) and of adenosine receptors with 8-phenyltheophylline (8PT), bath application of the P2X receptor agonist 3′-0-(4-benzoyl)ATP (BzATP) elicited a 40.5±16.0% (mean±S.E.M., n=8, P=0.039) increase in evoked EPSC amplitude and significantly reduced paired pulse facilitation of evoked EPSCs. This response to BzATP (with suramin and 8PT present) was completely blocked by prior application of Brilliant Blue G (200 nM or 2 μM), a P2X₇RS antagonist. In contrast, BzATP application with suramin and 8PT present did not alter miniature EPSC frequency or amplitude when action potentials were blocked with tetrodotoxin. These electrophysiological results suggest that P2X₇RS activation increases central excitatory transmitter release via presynaptic mechanisms, confirming previous indirect measures of enhanced transmitter release. We suggest that possible presynaptic mechanisms underlying enhancement of evoked transmitter release by P2X₇RS activation are modulation of action potential width or an increase in presynaptic terminal excitability, due to subthreshold membrane depolarization which increases the number of terminals releasing transmitter in response to stimulation.

Section snippets

Brain slice preparation and electrophysiological recording methods

Recordings were made from transverse brainstem slices containing the hypoglossal nucleus, prepared from 15 to 26 day-old Swiss CD1 mice (n=36) of either sex as described previously (Bellingham and Berger, 1996). Animal care and handling was approved by the University of Queensland animal ethics committee, and was in accordance with university and the National Health and Medical Research Council Australian code of practice for the care and use of animals for scientific purposes. All efforts were

Activation of multiple purinergic receptors by BzATP depresses evoked synaptic transmission

Previous studies have showed that P2X₇RS are several-fold more sensitive to activation by the ATP analog BzATP than by ATP (Surprenant et al., 1996; Lundy et al., 2002; Chessell et al., 1998; Hibell et al., 2001). Bath application of BzATP (30 μM) alone elicited a 37.6±8.3% (n=5, P=0.01) reduction in evoked EPSC amplitude in HMs (Fig. 1A, open bar and 1B1). Prolonged washout of up to an hour was able to reverse BzATP effects (Fig. 1B1), indicating that EPSC depression was not due to cytolytic

Discussion

Using electrophysiological recordings of evoked EPSCs, we have directly demonstrated that presynaptic P2X₇RS activation enhances evoked excitatory neurotransmitter release, confirming previous indirect measures of enhanced synaptic release following P2X₇RS activation (Deuchars et al., 2001; Sperlagh et al., 2002; Lundy et al., 2002). By comparing effects of P2X₇RS activation on evoked EPSCs to effects on miniature and spontaneous EPSCs, we also have established that P2X₇RS enhance transmitter

Acknowledgments

We thank Drs. Greg Funk, Jim Deuchars, David Knight and Refik Kanjhan for their valuable comments on the manuscript and other contributions to this work. The SV2 monoclonal antibody developed by Dr. K. M. Buckley was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA, USA. This work was supported by National Health and Medical Research Council (Australia),

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Therefore, the potential contribution of these subunits could be minimal; while the increments in P2X7 and P2X2 receptors appear to be the most important since these two subunits have been described as being prevalent in the plasma membrane (Chaumont et al., 2004; Murrell-Lagnado and Qureshi, 2008; Murrell-Lagnado and Robinson, 2013; Richler et al., 2011; Shrivastava et al., 2013). However, P2X7 receptors have been described as markers (as well as P2X4) for microglia activation (Anccasi et al., 2012; Avignone et al., 2008; Cavaliere et al., 2003; Choi et al., 2007; Doná et al., 2009) and are implicated in cell death (Anccasi et al., 2012; Cho et al., 2010; Choi et al., 2007; Ireland et al., 2004; León et al., 2008; Marcoli et al., 2008); while other reports have suggested contributions in cell survival (Amstrup and Novak, 2003; Gendron et al., 2003; Neary et al., 2003; Ortega et al., 2010). The role of P2X7R on microglia activation correlated with our data.
The most common cause of dementia is Alzheimer's disease. The etiology of the disease is unknown, although considerable evidence suggests a critical role for the soluble oligomers of amyloid beta peptide (Aβ). Because Aβ increases the expression of purinergic receptors (P2XRs) in vitro and in vivo, we studied the functional correlation between long-term exposure to Aβ and the ability of P2XRs to modulate network synaptic tone. We used electrophysiological recordings and Ca²⁺ microfluorimetry to assess the effects of chronic exposure (24 h) to Aβ oligomers (0.5 μM) together with known inhibitors of P2XRs, such as PPADS and apyrase on synaptic function. Changes in the expression of P2XR were quantified using RT-qPCR. We observed changes in the expression of P2X1R, P2X7R and an increase in P2X2R; and also in protein levels in PC12 cells (143%) and hippocampal neurons (120%) with Aβ. In parallel, the reduction on the frequency and amplitude of mEPSCs (72% and 35%, respectively) were prevented by P2XR inhibition using a low PPADS concentration. Additionally, the current amplitude and intracellular Ca²⁺ signals evoked by extracellular ATP were increased (70% and 75%, respectively), suggesting an over activation of purinergic neurotransmission in cells pre-treated with Aβ. Taken together, our findings suggest that Aβ disrupts the main components of synaptic transmission at both pre- and post-synaptic sites, and induces changes in the expression of key P2XRs, especially P2X2R; changing the neuromodulator function of the purinergic tone that could involve the P2X2R as a key factor for cytotoxic mechanisms. These results identify novel targets for the treatment of dementia and other diseases characterized by increased purinergic transmission.
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This compound reportedly has no effects on other P2 receptor subtypes and exhibits little or no activity against a wide array of other cell-surface receptors and ion channels (Nelson et al., 2006). Using superfused isolated nerve endings, we directly demonstrated that presynaptic P2X7 receptor activation enhances basal and [K+]e-evoked release of [3H]d-ASP, thereby confirming previous reports that stimulation of this receptor class augments vesicular GLU release in the rat and mice brainstem (Deuchars et al., 2001; Ireland et al., 2004). ATP interacting with several receptor subtypes, it is capable of triggering and maintaining the states of heightened sensory neuron excitability associated with persistent pain (Tsuda et al., 2009; Burnstock, 2009).
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P2X7-like receptor subunits enhance excitatory synaptic transmission at central synapses by presynaptic mechanisms

Abstract

Section snippets

Brain slice preparation and electrophysiological recording methods

Activation of multiple purinergic receptors by BzATP depresses evoked synaptic transmission

Discussion

Acknowledgments

Pharmacol Ther

Neuroscience

Neurosci Lett

Neuron

Eur J Pharmacol

FEBS Lett

J Neurosci Methods

Biophys J

Neuron

Neurosci Lett

J Auton Nerv Syst

Neuroscience

J Biol Chem

Brain Res

Lancet

Neuropharmacol

Cell Signal

Eur J Pharmacol

Trends Neurosci

J Neurosci

J Biol Chem

Neuron

Prog Neurobiol

Activation of presynaptic P2X7-like receptors depresses mossy fiber-CA3 synaptic transmission through p38 mitogen-activated protein kinase

J Neurosci

Determinants of the time course of facilitation at the granule cell to Purkinje cell synapse

J Neurosci

Oxidized ATP (oATP) attenuates proinflammatory signaling via P2 receptor-independent mechanisms

Br J Pharmacol

Presynaptic depression of excitatory synaptic inputs to rat hypoglossal motoneurons by muscarinic M2 receptors

J Neurophysiol

Excitatory actions of norepinephrine on multiple classes of hippocampal CA1 interneurons

J Neurosci

Molecular cloning and characterization of rat P2Y4 nucleotide receptor

Br J Pharmacol

P2X7, P2Y1 and P2Y2 receptors in rat alveolar macrophages

Br J Pharmacol

Concomitant blockade of P2X-receptors and ecto-nucleotidases by P2-receptor antagonists: functional consequences in rat vas deferens

Naunyn Schmiedebergs Arch Pharmacol

Cloned and transfected P2Y4 receptors: characterization of a suramin and PPADS-insensitive response to UTP

Br J Pharmacol

Properties of the pore-forming P2X7 purinoreceptor in mouse NTW8 microglial cells

Br J Pharmacol

Cloning of P2X5 and P2X6 receptors and the distribution and properties of an extended family of ATP-gated ion channels

J Neurosci

Neuronal P2X7 receptors are targeted to presynaptic terminals in the central and peripheral nervous systems

J Neurosci

P2X7 receptor-mediated release of excitatory amino acids from astrocytes

J Neurosci

P2X₇-like receptor subunits enhance excitatory synaptic transmission at central synapses by presynaptic mechanisms

Activation of presynaptic P2X₇-like receptors depresses mossy fiber-CA3 synaptic transmission through p38 mitogen-activated protein kinase

Cloning of P2X₅ and P2X₆ receptors and the distribution and properties of an extended family of ATP-gated ion channels

Neuronal P2X₇ receptors are targeted to presynaptic terminals in the central and peripheral nervous systems

P2X₇ receptor-mediated release of excitatory amino acids from astrocytes