Opposing role of synaptic and extrasynaptic NMDA receptors in regulation of the extracellular signal-regulated kinases (ERK) activity in cultured rat hippocampal neurons - PubMed (original) (raw)

Opposing role of synaptic and extrasynaptic NMDA receptors in regulation of the extracellular signal-regulated kinases (ERK) activity in cultured rat hippocampal neurons

Anton Ivanov et al. J Physiol. 2006.

Abstract

The extracellular signal-regulated kinases (ERK) signalling cascade is a key pathway that mediates the NMDA receptor (NMDAR)-dependent neuronal plasticity and survival. However, it is not clear yet how NMDARs regulate ERK activity. Stimulation of the NMDARs induces a complex modification of ERK that includes both ERK activation and inactivation and depends on particular experimental conditions. Here we show that there exists a differential restriction in the regulation of ERK activity that depends on the pool of NMDAR that was activated. The synaptic pool of NMDARs activates ERK whereas the extrasynaptic pool does not; on the contrary, it triggers a signalling pathway that results in the inactivation of ERK. As a result, simultaneous activation of both extrasynaptic and synaptic NMDAR using bath application of NMDA or glutamate (a typical protocol explored in the majority of studies) produced ERK activation that depended on the concentration of agonists and was always significantly weaker than those mediated by synaptic NMDARs. Since the activation of the extrasynaptic NMDA is attributed mainly to global release of glutamate occurring at pathological conditions including hypoxic/ischaemic insults, traumas and epileptic brain damage, the reported differential regulation of ERK cascade by NMDARs provides a unique mechanism for an early identification of the physiological and/or pathophysiological consequences of NMDAR activation. The negative regulation of the ERK activity might be one of the first signalling events determining brain injury and constitutes a putative target of new pharmacological applications.

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Figures

Figure 1

Figure 1. Activation of ERK by different subpopulations of NMDARs

A, example of voltage clamp whole cell recording of spontaneous excitatory postsynaptic currents (EPSCs) that reflect activity of the neuronal network during bath application of bicuculline and co-application of MK-801 1 min later. _V_h=−60 mV. B, neuron treatment with MK-801 in presence of bicuculline effectively inhibits synaptic NMDAR component of EPSCs. Superimposed traces illustrate spontaneous EPSCs recorded in the presence of bicuculline before (black) and after (grey) neuron incubation with MK-801. Plot shows MK-801-induced decrease of the amplitude of slow (NMDAR) component of EPSCs measured 100 ms after peak value. Mean data from 4 experiments, 5–7 events in each experiment. C, images of phosphorylated ERK1, 2 (P-ERK) and MAP2, in neurons activated via synaptic and extrasynaptic NMDARs. D, time course of ERK phosphorylation after activation of different populations of NMDARs. Abscissa indicates the time of treatment of cultures using mentioned protocols. Mean ±

s.e.m.

from 4 experiments. Bicuculline induced significantly different phosphorylation of ERK (P < 0.05) at all studied time points as compared with other experimental conditions.

Figure 2

Figure 2. Differential sensitivity of P-ERK to reduction of NMDA current through extrasynaptic and entire pool of NMDARs

A, currents induced by bath activation of 10 μ

m

NMDA before and after block of the synaptic NMDARs with MK-801 (entire NMDA and extrasynaptic NMDA, respectively). B, dose dependence of the inhibition of NMDA (10 μ

m

)-induced currents by

d

-AP5. n = 7. Example shows current recorded during application of 10 μ

m

NMDA and NMDA with 10 μ

m

D-AP5. C, inhibition of the NMDAR with 20 μ

md

-AP5 does not affect the NMDA-induced ERK phosphorylation. Neurons were incubated with indicated agonists and blockers for 5 min. Mean ±

s.e.m.

from 5 experiments.

Figure 3

Figure 3. ERK phosphorylation at different strengths of NMDAR activation

Phosphorylation of the ERK after NMDAR activation during 5 min with bicuculline, glutamate, different concentrations of NMDA and under different experimental conditions as indicated by (+) and (−). The basal external solution was as described in Methods. n = 4.

Figure 4

Figure 4. Activation of the extrasynaptic NMDAR induces dephosphorylation of P-ERK

A, scheme of the experiment. Horizontal bars indicate the duration of incubation of neurons with indicated drugs. B, mean data from 3 experiments showing that activation of the extrasynaptic pool of NMDAR induces dephosphorylation of P-ERK. C, ERK phosphorylation in neuronal cultures incubated during indicated times with bicuculline, bicuculline + DL-TBOA (50 μ

m

), and NMDA (10 μ

m

). Mean data from 4 experiments. Asterisks indicate values of ERK phosphorylation significantly different from others measured at the same time points (P < 0.01).

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