We initially examined the effects of mGluR2/3 activation on NMDAR-mediated field EPSPs (fEPSPNMDA) in the CA1 region. Application of the selective mGluR2/3 agonist LY 379268 (30nM) did not affect the amplitude of fEPSPNMDA (); however, there was an increase in the variance of the fEPSPNMDA response, suggesting that postsynaptic NMDAR function may be enhanced in spite of a reduction in presynaptic release of glutamate. To determine whether mGluR2/3 activation enhances postsynaptic NMDAR function, we examined the AMPA/NMDA ratio of excitatory postsynaptic currents (EPSCs) at Schaffer Collateral-CA1 synapses. Application of LY 379268 (30nM) resulted in a significant decrease of AMPA/NMDA EPSC ratio (p<0.05, ), suggesting an enhancement of postsynaptic NMDAR function. To more directly examine the effects of mGluR2/3 activation on NMDARs, we examined the actions of LY 379268 on acutely isolated CA1 pyramidal neurons.
Activation of group II mGluRs enhances postsynaptic NMDAR function.
We previously demonstrated that Gq- and Gs-coupled receptors potentiate NMDAR currents in isolated CA1 neurons via activation Src kinase and PKA, respectively19,20,21,22,23
. Given that mGluR2/3 couple to Gi/o and inhibit adenylyl cyclase activity14
, we anticipated that these receptors would inhibit NMDAR-mediated currents. To examine the direct effects of group II mGluRs on NMDARs, we determined the actions of LY 379268 on acutely isolated CA1 pyramidal neurons. These isolated neurons have a population of both extrasynaptic and synaptic NMDARs. Surprisingly, application of LY 379268 (10nM) to acutely isolated CA1 neurons potentiated NMDA-evoked currents, with a significant potentiation occurring after washout of LY 379268 (). The concentration of LY 379268 employed was chosen based on the 50% percent inhibitory concentration (IC50
) value of LY 379268 to displace 3
HLY341495, a group II selective antagonist radioligand, from native rat brain homogenates and recombinant human mGlu2 and mGlu3 receptor subtypes24
. The LY 379268-induced enhancement of NMDAR-mediated currents was blocked by co-application of the selective mGluR2/3 antagonist LY 341 495 (10nM) (). Conversely, co-application of the selective group I mGluR antagonist, MPEP hydrochloride, did not prevent the enhancement of NMDAR currents by LY 379268 (). These results confirm that the LY 379268-mediated potentiation of NMDAR currents was indeed mediated by mGlu2/3 receptors. Given that PKA promotes Ca2+
permeation through NMDARs and increases the amplitude of NMDA-evoked currents25
, it seems counterintuitive that inhibition of the PKA pathway via group II mGluRs would mediate the enhancement of NMDAR currents. Instead, we hypothesized that stimulation of group II mGluRs results in a promiscuous activation of Gq with a subsequent activation of PKC and/or Src leading to an enhancement of NMDAR currents19,20,22,23
. To test this hypothesis, we employed the Src(4058) peptide which mimics the unique domain of Src and prevents its interaction with the NADH dehydrogenase subunit 2 domain in the NMDAR complex26
. Thus, Src(4058) acts as an interfering peptide to prevent the regulation of NMDARs by endogenous Src26
. Including Src(4058) in the patch pipette prevented the enhancement of NMDAR currents by LY 379268 (). The LY 379268-induced enhancement of NMDAR currents was not, however, prevented by application of the PKC inhibitor, bisindolylmaleimide I (0.5 M, data not shown) suggesting that Src might be activated by an alternative mechanism. Using an approach similar to Gingrich et al
, we synthesized the Fyn interfering peptide, Fyn (3957) that corresponds to a region of the unique domain of Fyn. We previously showed that Fyn(3957) selectively blocks the potentiation of NMDAR currents by recombinant Fyn kinase but not by recombinant Src kinase23
. Application of Fyn(3957) inside the patch pipette failed to prevent the enhancement of NMDAR currents by LY 379268 () indicating that LY 379268 enhances Src but not Fyn activity to regulate NMDARs. To confirm these electrophysiological findings, we determined the relative activation of Src versus Fyn in hippocampal slices treated with or without LY 379268 (30nM). The mGluR2/3 agonist enhanced phosphorylation of Src at Y416 (), a site whose phosphorylation is required for activation this kinase27
. The LY 379268-mediated increase in Src Y416 phosphorylation was prevented by co-application of the mGluR2/3 antagonist LY 341 495 (). In contrast, LY 379268 failed to enhance the tyrosine phosphorylation of the analogous activation site of Fyn kinase, Y420 ()28
. These findings illustrate that Src, and not Fyn, regulates the mGluR2/3-mediated modulation of NMDAR currents in dissociated CA1 neurons.
Application of LY 379268 to acutely isolated CA1 neurons potentiates NMDAR currents.
The LY 379268-mediated increase of NMDA-evoked currents is mediated by group II metabotropic glutamate receptors.
mGluR2/3 activation recruits Src kinase to potentiate NMDAR currents in acutely isolated CA1 cells.
We previously reported that several GPCRs acting via Gq activate Src kinase to phosphorylate the GluN2A subunit of this receptor22,23
. In this regard, we employed applications of zinc (300 M) to selectively inhibit responses to heteromeric NMDARs containing two GluN2A subunits23,29
. The enhancement of NMDAR currents by LY 379268 was absent when GluN2A subunits were blocked with Zn2+
(). In contrast, the mGluR2/3-mediated modulation of NMDARs was still observed in the presence of the selective GluN2B antagonist, Ro 256981 (500nM) (). We also examined the potential phosphorylation of GluN2A and GluN2B subunits and found that applications of LY 379268 enhanced tyrosine phosphorylation of GluN2A but not GluN2B subunits (). Thus, activation of mGluR2/3 selectively potentiates GluN2A-containing NMDAR currents.
Stimulation of group II metabotropic glutamate receptors potentiates GluN2A-containing NMDAR currents.
Gs-dependent receptors enhance PKA and target Fyn kinase activation to enhance phosphorylation of NMDARs23
. Given that Fyn kinase was not activated by LY 379268 and that mGluR2/3 classically signal via inhibition of PKA, we initially confirmed that LY 379268 does indeed inhibit cAMP in treated hippocampal slices. Applications of LY 379268 reduced basal levels of cAMP in a concentration-dependent manner with an IC50
of 10.932nM ().
Group II metabotropic glutamate receptors inhibit cAMP formation and prevent PKA-mediated phosphorylation of Csk.
Src is strongly regulated in CA1 hippocampal neurons by C-terminal Src kinase (Csk), which phosphorylates Src on the C-terminal tyrosine Tyr527 and maintains it in an inactive conformation30
. Furthermore, the tyrosine kinase activity of Csk is increased by PKA-mediated phosphorylation31
, suggesting that a decrease in PKA activity might inhibit Csk and lead to a disinhibition of Src activity. To test this possibility, we first determined whether activation of mGluR2/3 inhibits Csk activity. Treatment of hippocampal membranes with LY379268 led to a significant reduction of Csk activity, as assessed by phosphorylation of Ser364 (). Furthermore, LY379268 treatment significantly reduced Csk-mediated phosphorylation of Src at its C-terminal regulatory tyrosine Y527 (). Decreased phosphorylation of Y527 on Src prevents the intramolecular interaction with the SH2 domain and leads to an open conformation of Src32,33
. Thus, activation of mGluR2/3 may activate Src through inhibition of its regulatory partner Csk. To determine whether inhibition of PKA may activate Src and occlude the effects of LY379268 on NMDAR currents, we applied a highly selective PKA inhibitor in the patch pipette and examined the modulation of NMDAR currents by LY 379268. Under this condition LY 379268 failed to enhance NMDAR currents () suggesting that Src is activated, at least in part, by a reduction in PKA activity.
Inhibition of PKA via group II metabotropic glutamate receptors activates Src kinase.