Potentiation Of NMDA Receptor Function Research Articles

Spinal neuronal NOS activation mediates sigma‐1 receptor‐induced mechanical and thermal hypersensitivity in mice: involvement of PKC‐dependent GluN1 phosphorylation

BACKGROUND AND PURPOSE We recently demonstrated that activation of the spinal sigma-1 receptor induces mechanical and thermal hypersensitivity via calcium-dependent second messenger cascades and phosphorylation of the spinal NMDA receptor GluN1 subunit (pGluN1). Here we examined the role of NO in this process, as it plays a critical role in PKC-mediated calcium signalling and the potentiation of NMDA receptor function. EXPERIMENTAL APPROACH The effects of intrathecal (i.t.) pretreatment with nNOS inhibitors on PRE084 (sigma-1 receptor agonist)-induced pain were assessed in mice by use of mechanical allodynia and thermal hyperalgesia tests. Western blot analysis, immunoprecipitation and immunohistochemical techniques were used to determine effects of these treatments on spinal pGluN1-immunoreactive (ir) cells, whether PRE084 induces a time-dependent modification of nNOS activity in the dorsal horn, and if any changes in nNOS activity can be blocked by sigma-1 receptor, calcineurin or soluble guanylyl cyclase (sGC) inhibitors. KEY RESULTS PRE084, injected i.t., induced mechanical and thermal hypersensitivity, and increased the number of PKC- and PKA-dependent pGluN1-ir cells in spinal cord. This PRE084-induced hypersensitivity and increase in PKC-dependent pGluN1 expression were blocked by pretreatment with N(G) -nitro-L-arginine methyl ester (L-NAME) or 7-nitroindazole (7-NI). PRE084 also time-dependently decreased the ratio of phosphorylated nNOS (pnNOS) to nNOS expression and the number of spinal pnNOS-ir cells. This decrease in pnNOS was prevented by BD1047, a sigma-1 receptor antagonist and cyclosporin A, a calcineurin inhibitor, but not by a sGC inhibitor. CONCLUSIONS AND IMPLICATIONS Spinal sigma-1 receptor-induced sensitization is mediated by an increase in nNOS activity, which is associated with an NO-induced increase in PKC-dependent pGluN1 expression.

Sigma-1 receptor-induced increase in murine spinal NR1 phosphorylation is mediated by the PKCα and ɛ, but not the PKCζ, isoforms

Our previous studies have demonstrated that intrathecal (i.t.) administration of a sigma-1 receptor agonist facilitated peripheral nociception via calcium-dependent second messenger cascades including protein kinase C (PKC). We also showed that activation of spinal sigma-1 receptors increased the phosphorylation of the NMDA receptor NR1 subunit (pNR1) in the spinal cord dorsal horn, which resulted in the potentiation of NMDA receptor function. The present study was designed to examine the effect of different PKC isoform inhibitors on sigma-1 receptor-mediated pain facilitation and increased spinal pNR1 expression in mice. The intrathecal injection of the sigma-1 receptor agonist, PRE-084 (PRE, 3 nmol/5 μl) increased the frequency of paw withdrawal responses to mechanical stimuli (0.6 g) and the number of spinal pNR1-immunoreactive (ir) cells. Intrathecal pretreatment with inhibitors (Go6976, PKCɛV1–2 or PKC ζpseudosubstrate) of the PKCα, ɛ or ζ isoforms significantly reduced the PRE-induced pain facilitatory effect. On the other hand, the PRE-induced increase in the number of spinal pNR1-ir neurons was only blocked by inhibitors of the PKCα and PKCɛ isoforms, but not the PKCζ isoform. These findings demonstrate that the sigma-1 receptor-induced increase in spinal pNR1 expression is mediated by the PKCα and PKCɛ isoforms, which in turn contribute to the pain facilitation phenomenon. Conversely, the sigma-1 receptor activation of the PKCζ isoform appears to be involved in a pain signaling pathway that is independent of spinal pNR1 modulation.

Functional interactions between presynaptic NMDA receptors and metabotropic glutamate receptors co‐expressed on rat and human noradrenergic terminals

Electrophysiological studies described potentiation of NMDA receptor function by metabotropic glutamate receptors (mGluRs) of group I occurring postsynaptically. Since release-enhancing NMDA receptors exist on noradrenergic terminals and group I mGluRs have recently been identified on these nerve endings, we have investigated if NMDA receptor-mGluR interactions also can occur at the presynaptic level. Rat hippocampus and human neocortex synaptosomes were labelled with [(3)H]noradrenaline and superfused with mGluR agonists and antagonists. NMDA-evoked [(3)H]noradrenaline release was produced by removal of external Mg(2+) or by simultaneous application of NMDA and AMPA in Mg(2+)-containing solutions. The mGluR1/5 agonist 3,5-DHPG, inactive on its own, potentiated both the release of [(3)H]noradrenaline elicited by AMPA/NMDA/glycine and that evoked by NMDA/glycine following Mg(2+) removal. The effect of 3,5-DHPG on the AMPA/NMDA/glycine-induced release was insensitive to the mGluR1 antagonist CPCCOEt, but it was abolished by the mGluR5 antagonist MPEP; moreover, it was potentiated by the mGluR5 positive allosteric modulator DFB. When NMDA receptors were activated by Mg(2+) removal, both mGluR5 and mGluR1 contributed to the evoked release, the mGluR-mediated release being blocked only by CPCCOEt and MPEP in combination. Experiments with human neocortex synaptosomes show NMDA receptor-mGluR interactions qualitatively similar to those observed in rodents. Group I mGluRs, both of the mGluR1 and mGluR5 subtypes, co-localize with NMDA receptors on noradrenergic terminals of rat hippocampus and human neocortex. Depending on the mode of activation, NMDA receptors exert differential permissive roles on the activation of presynaptic mGluR1 and mGluR5.

Glycine Transporter 1 Inhibitors and Modulation of NMDA Receptor-Mediated Excitatory Neurotransmission

In the central nervous system, glutamate is essential for a proper synaptic communication in neuronal networks supporting critical behavioral activities such as learning and memory. Dysfunction of glutamatergic excitatory neurotransmission has been implicated in numerous neurological and pyschiatric disorders and a growing body of research suggests that potentiation of NMDA receptor function may represent a novel approach for the treatment of schizophrenia. An actively pursued strategy to potentiate NMDA receptor function is to increase synaptic levels of the neurotransmitter glycine by blocking the glycine transporter type 1 (GlyT1). Since glycine acts as a co-agonist at the NMDA receptor, this approach could enhance the effectiveness of normal NMDA receptor-mediated glutamatergic neurotransmission. Recent research on the physiology of this uptake system as well as on the development and preclinical testing of novel GlyT1 inhibitors have greatly enhanced our knowledge of the role of this transporter in the modulation of NMDA receptor activity and suggested that this approach may be feasible. Clinical studies with novel glycine reuptake inhibitors will provide critical information regarding the validity of this therapeutic concept for the treatment of schizophrenia and other disorders associated with NMDA receptor hypofunction.

Involvement of a Dysfunctional Dopamine-D1/N-Methyl-d-aspartate-NR1 and Ca2+/Calmodulin-Dependent Protein Kinase II Pathway in the Impairment of Latent Learning in a Model of Schizophrenia Induced by Phencyclidine

Continuous ingestion of phencyclidine (PCP) in humans produces long-lasting schizophrenic-like cognitive dysfunction. Although a malfunction of dopaminergic and/or glutamatergic neurotransmission is implicated in the etiology of schizophrenia, involvement of the dopaminergic-glutamatergic neurotransmission in the cognitive dysfunction induced by repeated PCP treatment is minor. We demonstrated that mice treated with PCP (10 mg/kg/day s.c.) for 14 days displayed an impairment of latent learning in a water-finding task and of learning-associated phosphorylation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and NR1 in the prefrontal cortex even after drug withdrawal. The infusion of a CaMKII inhibitor and NR1 antisense oligonucleotide into the prefrontal cortex produced an impairment of latent learning and decrease of learning-associated phosphorylation of CaMKII, which were observed in the PCP-treated mice. Exogenous NMDA-induced CaMKII activation was not observed in slices of the prefrontal cortex prepared from mice treated repeatedly with PCP. The potentiation of NMDA receptor function by the infusion of glycine into the prefrontal cortex ameliorated these impairments in mice treated repeatedly with PCP. The high potassium-stimulated release of dopamine from the prefrontal cortex was less extensive in the PCP-treated than saline-treated mice. The infusion of a dopamine-D1 receptor agonist into the prefrontal cortex attenuated the impairment of latent learning and decrease of learning-associated NR1 phosphorylation in the PCP-treated mice, suggesting a functional linkage between glutamatergic and dopaminergic signaling. These findings indicate that repeated PCP treatment impairs latent learning through a prefrontal cortical dysfunction of NMDA-CaMKII signaling, which is associated with dopaminergic hypofunction.

Modulation of NMDA receptors by AKT kinase

The aim of the present work was to assess whether Akt modulates NMDA receptor function in cerebellar neurons in culture. Forskolin increases cAMP and activates Akt and NMDA receptors. In neurons treated with forskolin, intracellular calcium increased to 296 ± 38% and this was completely prevented by inhibition of Akt. This indicates that, in these neurons, cAMP modulates NMDA receptors via Epac and Akt. Brain derived neurotrophic factor (BDNF) increases phosphorylation (and activity) of Akt to 350 ± 60% of basal and also potentiates the increase of calcium and in cGMP induced by NMDA. BDNF-induced potentiation of NMDA receptor function is completely prevented by inhibition of PI3 kinase or of Akt. This indicates that BDNF modulates NMDA receptor function via PI3 kinase and Akt. Activation of NMDA receptors also leads to phosphorylation and activation of Akt which, in turn, potentiates NMDA receptor activation. The results reported indicate that when Akt activity increases the activation of NMDA receptors by its agonists also increases. Akt may play important roles in the modulation of NMDA receptor responses by other neurotransmitters and modulators and in the adaptation of NMDA receptor function to the physiological environmental conditions.

Activation of NMDA receptors and L-type voltage-gated calcium channels mediates enhanced formation of Fyn–PSD95–NR2A complex after transient brain ischemia

Recent studies have indicated that tyrosine phosphorylation of NMDA receptor subunit 2A (NR2A) by Src family kinases (Src, Fyn, etc.) up-regulates NMDA receptors activity and postsynaptic density protein 95 kDa (PSD95) may mediate the regulation. To investigate whether the above processes are involved in brain ischemia-induced enhancement of NMDA receptors function, we examined the effects of transient (15 min) brain ischemia followed by reperfusion on interactions involving Fyn, NR2A and PSD95 in rat hippocampus by co-immunoprecipitation. Transient brain ischemia was induced by the method of four-vessel occlusion in Sprague–Dawley rats. Association between Fyn and NR2A increased immediately after brain ischemia and the increase was maintained for at least 24 h during followed reperfusion, up to about 1.7–1.8-fold relative to sham-groups. The 15-min reperfusion after brain ischemia induced enhanced co-immunoprecipitation of PSD95, Fyn and NR2A with one another. The associations of PSD95 with Fyn and NR2A increased at 0–24 h, 0–1 h of reperfusion, up to 6.9- and 2.1-fold relative to sham groups, respectively. Inhibiting activation of NMDA receptors or L-type voltage-gated calcium channels (L-VGCC) by ketamine or nifedipine attenuated the above increases of associations. These results suggest that stimulation of NMDA receptors and L-VGCC facilitates formation of a ternary complex: Fyn–PSD95–NR2A during transient brain ischemia followed by reperfusion, which may result in potentiation of NMDA receptor function and contribute to ischemic neuronal cell death.

Potentiation of NMDA receptor function through somatostatin release: a possible mechanism for the cognition-enhancing activity of GABA B receptor antagonists

CGP 36742 is a weak GABA B receptor antagonist. However, it improves cognitive performances at low doses; it blocks GABA B receptors potently and selectively on somatostatinergic terminals; it prevents kynurenate from antagonising NMDA-induced release of noradrenaline from rat brain slices potently. We here investigated whether and how somatostatin plays a role in the CGP 36742 activity. CGP 36742 increased the somatostatin-like immunoreactivity (SRIF-LI) release from hippocampal slices exposed to NMDA. In the kynurenate test with rat hippocampal slices SRIF-14 mimicked the effect of CGP 36742. CGP 36742 lost its activity in rats whose somatostatin content had been depleted with cysteamine. Exogenous SRIF-14 reverted kynurenate antagonism in somatostatin-depleted slices. L362855, an sst 5 receptor agonist, but not the selective sst 1–sst 4 agonists, L797591, L779976, L796778 and L803087, displayed activity in the kynurenate test. The effects of CGP 36742, SRIF-14 and L362855 were antagonised by the sst 5-preferring antagonist BIM-23056. The protein kinase C inhibitor GF 109203X prevented the reversal of the kynurenate antagonism by CGP 36742 or SRIF-14. In conclusion, by selectively blocking GABA B receptors on somatostatinergic terminals, CGP 36742 may disinhibit somatostatin release; the consequent activation of sst 5 receptors would potentiate the function of NMDA receptors coexisting with sst 5 receptors on noradrenergic neurons.

Protein kinase C activation induces tyrosine phosphorylation of the NR2A and NR2B subunits of the NMDA receptor.

The N-methyl-D-aspartate receptor (NMDAR) is an ionotropic glutamate receptor, which plays crucial roles in synaptic plasticity and development. We have recently shown that potentiation of NMDA receptor function by protein kinase C (PKC) appears to be mediated via activation of non-receptor tyrosine kinases. The aim of this study was to test whether this effect could be mediated by direct tyrosine phosphorylation of the NR2A or NR2B subunits of the receptor. Following treatment of rat hippocampal CA1 mini-slices with 500 nM phorbol 12-myristate 13-acetate (PMA) for 15 min, samples were homogenized, immunoprecipitated with anti-NR2A or NR2B antibodies and the resulting pellets subjected to Western blotting with antiphosphotyrosine antibody. An increase in tyrosine phosphorylation of both NR2A (76 +/- 11% above control) and NR2B (41 +/- 11%) was observed. This increase was blocked by pretreatment with the selective PKC inhibitor chelerythrine, with the tyrosine kinase inhibitor Lavendustin A or with the Src family tyrosine kinase inhibitor PP2. PMA treatment also produced an increase in the phosphorylation of serine 890 on the NR1 subunit, a known PKC site, at 5 min with phosphorylation returning to near basal levels by 10 min while tyrosine phosphorylation of NR2A and NR2B was sustained for up to 15 min. These results suggest that the modulation of NMDA receptor function seen with PKC activation may be the result of tyrosine phosphorylation of NR2A and/or NR2B.

Potentiation of NMDA Receptor Function by the Serine Protease Thrombin

Although serine proteases and their receptors are best known for their role in blood coagulation and fibrinolysis, the CNS expresses many components of an extracellular protease signaling system including the protease-activated receptor-1 (PAR1), for which thrombin is the most effective activator. In this report we show that activation of PAR1 potentiates hippocampal NMDA receptor responses in CA1 pyramidal cells by 2.07 +/- 0.27-fold (mean +/- SEM). Potentiation of neuronal NMDA receptor responses by thrombin can be blocked by thrombin and a protein kinase inhibitor, and the effects of thrombin can be mimicked by a peptide agonist (SFLLRN) that activates PAR1. Potentiation of the NMDA receptor by thrombin in hippocampal neurons is significantly attenuated in mice lacking PAR1. Although high concentrations of thrombin can directly cleave both native and recombinant NR1 subunits, the thrombin-induced potentiation we observe is independent of NMDA receptor cleavage. Activation of recombinant PAR1 also potentiates recombinant NR1/NR2A (1.7 +/- 0.06-fold) and NR1/NR2B (1.41 +/- 0.11-fold) receptor function but not NR1/NR2C or NR1/NR2D receptor responses. PAR1-mediated potentiation of recombinant NR1/NR2A receptors occurred after activation with as little as 300 pm thrombin. These data raise the intriguing possibility that potentiation of neuronal NMDA receptor function after entry of thrombin or other serine proteases into brain parenchyma during intracerebral hemorrhage or extravasation of plasma proteins during blood-brain barrier breakdown may exacerbate glutamate-mediated cell death and possibly participate in post-traumatic seizure. Furthermore, the ability of neuronal protease signaling to control NMDA receptor function may also have roles in normal brain development.

Src Potentiation of NMDA Receptors in Hippocampal and Spinal Neurons Is Not Mediated by Reducing Zinc Inhibition

The protein-tyrosine kinase Src is known to potentiate the function of NMDA receptors, which is necessary for the induction of long-term potentiation in the hippocampus. With recombinant receptors composed of NR1-1a/NR2A or NR1-1a/2B subunits, Src reduces voltage-independent inhibition by the divalent cation Zn2+. Thereby the function of recombinant NMDA receptors is potentiated by Src only when the Zn2+ level is sufficient to cause tonic inhibition. Here we investigated whether the Src-induced potentiation of NMDA receptor function in neurons is caused by reducing voltage-independent Zn2+ inhibition. Whereas chelating extracellular Zn2+ blocked the Src-induced potentiation of NR1-1a/2A receptors, we found that Zn2+ chelation did not affect the potentiation of NMDA receptor (NMDAR) currents by Src applied into hippocampal CA1 or CA3 neurons. Moreover, Src did not alter the Zn2+ concentration-inhibition relationship for NMDAR currents in CA1 or CA3 neurons. Also, chelating extracellular Zn2+ did not prevent the upregulation of NMDA single-channel activity by endogenous Src in membrane patches from spinal dorsal horn neurons. Taking these results together we conclude that Src-induced potentiation of NMDAR currents is not mediated by reducing Zn2+ inhibition in hippocampal and dorsal horn neurons.