Basal adenosine modulates the functional properties of AMPA receptors in mouse hippocampal neurons through the activation of A1R A2AR and A3R

Abstract

Adenosine is a widespread neuromodulator within the CNS and its extracellular level is increased during hypoxia or intense synaptic activity, modulating pre- and postsynaptic sites. We studied the neuromodulatory action of adenosine on glutamatergic currents in the hippocampus, showing that activation of multiple adenosine receptors (ARs) by basal adenosine impacts postsynaptic site. Specifically, the stimulation of both A1R and A3R reduces AMPA currents, while A2AR has an opposite potentiating effect. The effect of ARs stimulation on glutamatergic currents in hippocampal cultures was investigated using pharmacological and genetic approaches. A3R inhibition by MRS1523 increased GluR1-Ser845 phosphorylation and potentiated AMPA current amplitude, increasing the apparent affinity for the agonist. A similar effect was observed blocking A1R with DPCPX or by genetic deletion of either A3R or A1R. Conversely, impairment of A2AR reduced AMPA currents, and decreased agonist sensitivity. Consistently, in hippocampal slices, ARs activation by AR agonist NECA modulated glutamatergic current amplitude evoked by AMPA application or afferent fiber stimulation. Opposite effects of AR subtypes stimulation are likely associated to changes in GluR1 phosphorylation and represent a novel mechanism of physiological modulation of glutamatergic transmission by adenosine, likely acting in normal conditions in the brain, depending on the level of extracellular adenosine and the distribution of AR subtypes.

Keywords: A3R; AMPA receptors; adenosine receptors; hippocampal neurons; neuronal modulation.

FIGURE 1

Tonic activation of adenosine receptors modulates AMPA currents in mouse hippocampal neurons. (A) time course and sample traces of AMPA current (AMPA 10 μM, CTZ 25 μM) potentiation induced by acute treatment with Adenosine deaminase (ADA, 1 U/ml, n = 5; p < 0.05) in cultured hippocampal neurons. (B) Left, time course of AMPA current modulation by acute treatment with adenosine receptors antagonists blocking specifically: A₃R (MRS1523, 100 nM, n = 5, purple), A₃R + A₁R (MRS1523 + DPCPX, n = 5, green) or A_2AR (SCH58261 30 nM, n = 5, orange). Application of all drugs starts at t = 0, gray bar. Right, sample traces of AMPA currents in control (black) and in the presence of MRS1523 (purple) or SCH58261 (orange).

FIGURE 2

Basal activation of AR subtypes changes the apparent affinity for AMPA. (A) AMPA dose response curves (+ CTZ 25 μM) obtained in cultured hippocampal neurons in control (black, n = 32), in the presence of A₃R antagonist MRS1523 (100 nM, n = 11, purple) and in neurons from A₃R KO mice (n = 11, magenta). Inserts: sample traces representing current responses to 1, 10, and 100 μM AMPA (+ CTZ 25 μM) in WT and A₃R KO hippocampal neurons. (B) AMPA dose response curves (+ CTZ 25 μM) obtained in cultured hippocampal neurons in the presence of A_2AR antagonist SCH58261 (30 nM, n = 9, orange) and in neurons from A_2AR KO mice (n = 12; red; control condition in black). Inserts: representative current responses to 1, 10, and 100 μM AMPA (+ CTZ 25 μM) in A_2AR KO hippocampal neurons.

FIGURE 3

AMPARs function modulation requires neuronal A₃R activation. (A) Reduction of AMPA current amplitude induced by the application of A₃R agonist 2-Cl-IBMECA in cultured hippocampal neurons treated with Adenosine deaminase (1 U/ml, 1 h preincubation + perfusion); bar chart representing the effect observed in WT cultures (black; n = 12); or A₃R KO cultures (magenta; n = 13); gray column represents the effect of 2-Cl-IBMECA in A₃R KO cultures in the presence of WT glial cells (n = 12). (B) Sample traces showing the effect of 2-Cl-IBMECA on AMPA currents in hippocampal neurons from WT (top) and A₃R KO (bottom) mice, co-cultured with WT glial cells. ^∗∗p < 0.01.

FIGURE 4

A₃R dependent AMPARs modulation requires GluR1 dephosphorylation. (A) Time course of the effect of the A₃R agonist 2-Cl-IBMECA on AMPA current amplitude in the continuous presence of ADA (1 U/ml, 1 h preincubation) in control condition (black, n = 12) and after substitution of intracellular ATP with the thiophosphorylating agent ATPγS (n = 6). (B) Bar chart and sample traces displaying mean AMPA current potentiation induced by A₃R antagonist MRS1523 in control and in the presence of PKA blocker KT5720 (n = 6). (C) Representative immunoblot and quantitative western blot analysis of the effect of MRS1523 on the level of basal Glur1 Ser845 phosphorylation (n = 13). ^∗p < 0.05, ^∗∗p < 0.01.

FIGURE 5

N-ethyl 1-5′ carboxamido adenosine (NECA) modulates postsynaptic AMPA receptors through multiple ARs. (A) Time course and representative traces of AMPA (100 μM plus CTZ 25 μM) current amplitude reduction induced by NECA (t = 25 min) (100 nM) in hippocampal CA1 neurons (n = 6; p < 0.01). (B,C) Time course and bar chart (at t = 25 min) of the effect of NECA in the presence of combination of ARs antagonists: DPCPX (100 nM) plus MRS1523 (100 nM) (gray; n = 6; p < 0.01); SCH58261 (30 nM) plus MRS1523 (dark green; n = 6; p < 0.05); DPCPX plus SCH58261 (green; n = 5; p < 0.01). ^∗p < 0.05, ^∗∗p < 0.01, ^#p < 0.05, ^##p < 0.01.

FIGURE 6

Modulation of EPSC amplitude by NECA depends on A₃R. (A) Left, time course of EPSC depression induced by adenosine receptor agonist NECA (100 nM) in hippocampal CA1 neurons in from acute slices in control conditions (black; n = 7; p < 0.05 at t = 25 min), in the presence of A₃R antagonist MRS1523 (100 nM) (purple; n = 7; p < 0.05 respect to control and NECA alone) and in the presence of DPCPX + SCH58261 + MRS1523 (gray, n = 7). Right, sample EPCSs recorded in a CA1 pyramidal neuron in control and following NECA application. (B) Bar chart showing the effect of NECA (100 nM) alone (black), in the presence of the A₃R antagonist (MRS1523 100 nM, purple) and in the presence of all three blockers (DPCPX + SCH58261+MRS1523 gray, n = 7). ^∗p < 0.05, ^∗∗p < 0.01, ^#p < 0.05.