NR2B Signaling Research Articles

Focal Adhesion Kinase Inhibition Ameliorates Burn Injury-Induced Chronic Pain in Rats.

Burn injury-induced pain (BIP) is a significant global health concern, affecting diverse populations including children, military veterans, and accident victims. Current pharmacotherapeutics for the management of BIP are associated with severe side effects including drug addiction, respiratory depression, sedation, and constipation posing significant barrier to their clinical utility. In the present study, we have investigated the potential role of focal adhesion kinase (p-FAK) for the very first time in BIP and elucidated the associated underlying mechanisms. Defactinib (DFT), a potent p-FAK inhibitor, administered at doses of 5, 10, and 20mg/kg via intraperitoneal injection, demonstrates significant efficacy in reducing both evoked and spontaneous pain without causing addiction or other central nervous system toxicities. Burn injury triggers p-FAK-mediated phosphorylation of Erk1/2 and NR2B signaling in the DRG, resulting in heightened hypersensitivity through microglial activation, neuropeptide release, and elevated proinflammatory cytokines. Defactinib (DFT) counteracts these effects by reducing NR2B upregulation, lowering substance P levels, inhibiting microglial activation, and restoring IL-10 levels while leaving CGRP levels unchanged. These findings provide valuable insights into the pivotal role of p-FAK in regulating BIP and highlight the potential for developing novel therapeutics for burn injury-induced pain with minimal side effects.

Sida cordifolia L. attenuates behavioral hypersensitivity by interfering with KIF17-NR2B signaling in rat model of neuropathic pain

Ethnopharmacological relevanceSida cordifolia L., a perennial subshrub belonging to the Malvaceae family, holds noteworthy significance in the Indian Ayurvedic System and global texts. Roots of this plant are reported to be useful in neurodegenerative disorders, facial paralysis, and treating several neuropathic pain conditions such as neuralgia, and sciatica. However, despite these claims, there remains a dearth of experimental evidence showcasing the effectiveness of Sida cordifolia L. roots in mitigating neuropathic pain. Aim of the studyThe primary objective of this study was to assess the analgesic properties of the whole extract (SCE) obtained from the roots of Sida cordifolia L., as well as its aqueous fraction (SAF) in rat model of chronic constriction injury (CCI)-induced neuropathic pain. Furthermore, in-depth phytochemical and molecular biology studies were conducted to identify the potential phytoconstituents and unveil the underlying mechanisms of action. Material and methodsDCM: Methanol (1:1) was used to extract the roots of Sida cordifolia L. to get whole extract (SCE) and was subjected to phytochemical investigations including LC-MS analysis. Analgesic potential of SCE was evaluated in chronic constriction injury (CCI) model of neuropathic pain in rats followed by its bioactivity guided fractionation using in-vitro anti-inflammatory assay and assessment of most potent fraction (SAF) in in-vivo pain model. We have also performed the detailed phytochemical and molecular biology investigations to delineate the mechanism of action of Sida cordifolia root extract. ResultsChronic constriction injury leads to significant decrease in paw withdrawal threshold and paw withdrawal latency indicating development of hypersensitivity in rodents. Treatment with SCE and its most potent aqueous fraction (SAF) leads to significant and dose-dependent reduction in pain-like behavior of nerve injured rats. Pro-inflammatory cytokines (TNF-α, IL-1β), glia cell markers (Iba1, ICAM1), neuropeptides (CGRP and Substance P), KIF-17 and NR2B expressions were found to be significantly upregulated in DRG and spinal cord of nerve injured rats. Treatment with SCE and SAF suppressed oxido-inflammatory cascade along with attenuation of KIF-17 mediated NR2B trafficking and neuroinflammation in DRG and spinal tissues of neuropathic rats. HPTLC and HR-MS analysis suggest betaine as major constituent in SAF which along with other phytoconstituents. ConclusionsBoth the whole extract (SCE) and the aqueous fraction (SAF) demonstrate a significant reduction in mechanical and thermal hypersensitivity by inhibiting KIF-17 mediated NR2B signaling in nerve injured rats and may be used as a potential alternative for the treatment of chronic pain. Our findings support the use of roots of Sida cordifolia L. in neuropathic pain conditions as acclaimed by its traditional use.

Saffron carotenoids reversed the UCMS-induced depression and anxiety in rats: Behavioral and biochemical parameters, and hippocampal BDNF/ERK/CREB and NR2B signaling markers

BackgroundDepression is a debilitating condition that affects the mind and the individual's body. The improving effects of saffron on depression and anxiety have long been discussed, with limited information about the molecular mechanism of action. Hypothesis/PurposeInvestigating the effect of saffron carotenoids, Crocin and Crocetin, on depression and anxiety in rats by emphasizing some signaling pathways involved. Study DesignDepression and anxiety were induced in rats via unpredictable chronic mild stress (UCMS). Then different rat groups were treated with Crocin, Crocetin, Fluoxetine, and vehicle. Behavioral tests were done before and after treatment. MethodsThe serum Serotonin and Corticosterone and the expression of some hippocampal signaling proteins were studied. Furthermore, bioinformatics tools were used to predict the interactions of Crocin/ Crocetin with the Serotonin transporter and NMDA receptor subunit NR2B. Then, the patch-clamp was used to study the interaction of Crocetin with the NMDA receptor. ResultsVarious behavioral tests confirmed the induction of depression and the improvement of depression by these natural carotenoids. In addition, Crocin/ Crocetin significantly increased the decreased serum Serotonin and reduced the increased serum Corticosterone in the depressed groups. They also increased or caused a trend of increase in the CREB, ERK, BAD, BDNF, p11, and 5-HT1B expression in the hippocampus of the depressed groups. In addition, there were an increase or a trend in p-CREB/CREB, p-ERK1/2 /ERK1/2, and p-BAD/BAD ratios in the Crocin/ Crocetin treated depressed groups. However, the NR2B and FOXO3a expression showed a trend of decrease in depressed groups after treatment. The bioinformatics data indicated that Crocin/ Crocetin could bind to the Serotonin transporter (SLC6A4) and NR2B subunit of the NMDA receptor. Both carotenoids bind to the same site as Fluoxetine in the SLC6A4. However, they bound to different sites on the NR2B. So, Crocetin binds to NR2B at the same site as Ifenprodil. But Crocin bound to another site. The whole cell patch-clamp recording on the normal rat hippocampus revealed a significant decrease in the NMDA peak amplitude after Crocetin treatment, indicating its inhibitory effect on this receptor. ConclusionThe antidepressant activities of Crocin/ Crocetin are possibly due to their effects on Serotonin and Corticosterone serum concentrations, NR2B expression, and the downstream signaling pathways. Furthermore, these natural carotenoids, like Fluoxetine, induced an increasing tendency in p11 and 5HT1B in depressed rats.

Systemic Inflammation Increases the Susceptibility to Levodopa-Induced Dyskinesia in 6-OHDA Lesioned Rats by Targeting the NR2B-Medicated PKC/MEK/ERK Pathway.

Background: The long-term administration of levodopa (L-dopa), the gold-standard treatment for Parkinson's disease (PD), is irreparably associated with L-dopa-induced dyskinesia (LID), which dramatically affects the quality of life of patients. However, the underlying molecular mechanisms of how LID exacerbates remain unknown. Neuroinflammation in the striatum plays an active role in LID. These findings prompt an investigation of non-neuronal mechanisms of LID. This study will examine the effects of systemic inflammation in the development and progression of LID.Methods: To evaluate the possible influence of systemic inflammation in the appearance of LID, the PD rats received an intraperitoneal (IP) injection of various concentrations of lipopolysaccharides (LPS, 1, 2, and 5 mg/kg) or saline. One day later, these PD rats started to receive daily treatment with L-dopa (6 mg/kg) along with benserazide (6 mg/kg) or saline for 21 days, and dyskinesia was evaluated at several time points. Moreover, the activation of microglia and astrocytes and the molecular changes in NR2B and mGLUR5 signaling pathways were measured.Results: We found that systemic inflammatory stimulation with LPS exacerbated the intensity of abnormal involuntary movements (AIMs) induced by L-dopa treatment in 6-hydroxydopamine (6-OHDA) lesioned rats. The LPS injection activated the gliocytes and increased the levels of proinflammatory cytokines in the striatum in LID rats. The PD rats that received the LPS injection showed the overexpression of p-NR2B and NR2B, as well as activated PKC/MEK/ERK and NF-κB signal pathways in response to the L-dopa administration. On the contrary, clodronate-encapsulated liposomes (Clo-lipo), which could suppress the inflammatory response induced by peripheral LPS injection, improved behavioral dysfunction, inhibited neuroinflammation, prevented NR2B overexpression, and decreased the phosphorylation of PKC/MEK/ERK and NF-κB signaling pathways.Conclusion: This study suggests that systemic inflammation, by exacerbating preexisting neuroinflammation and facilitating NR2B subunit activity, may play a crucial role in the development of LID. The administration of Clo-lipo restores the effects of LPS and decreases the susceptibility to LID in 6-OHDA lesioned rats.

Neuroprotective Effects of Co-Administration of Selegiline with Piracetam on Cognitive Impairment: Involvement of NR2B, NR1 and Bax Signaling Pathway

Neuroprotective Effects of Co-Administration of Selegiline with Piracetam on Cognitive Impairment: Involvement of NR2B, NR1 and Bax Signaling Pathway

S-nitrosylation of Src by NR2B-nNOS signal causes Src activation and NR2B tyrosine phosphorylation in levodopa-induced dyskinetic rat model.

Abnormality in Src PSD-95 NR2B signaling complex assemble occurs in levodopa-induced dyskinesia (LID). N-methyl-D-aspartate receptor (NMDAR) subunit NR2B tyrosine phosphorylation mediated by Src family protein tyrosine kinases is closely associated with dyskinesia. Src autophosphorylation (p-Src) is an important part of Src-catalyzed phosphorylation of NR2B. In addition, the neuronal nitric oxide synthase (nNOS)-derived NO (nNOS/NO) signal which was also involved in dyskinesia recently was proved to participate in the regulation of Src function. Yet, the detailed signal mechanism about the interactions of NR2B, nNOS, and Src is still unknown. In the present study, we investigated the influences of nNOS on Src activation and NR2B tyrosine phosphorylation in dyskinetic rat model by immunoblotting and immunoprecipitation. The results demonstrated that chronic levodopa treatment resulted in downregulation of p-nNOS-S847, one marker of nNOS overactivation. Coinstantaneously, the S-nitrosylation (SNO-Src) and autophosphorylation (p-Src) of Src and NR2B tyrosine phosphorylation were upregulated in dyskinetic rat model. Conversely, administration of 7-NI, one nNOS inhibitor, reversed all these effects of levodopa treatment. Besides, NR2B-containing NMDAR (NR2B/NMDAR) antagonist CP-101,606 could upregulate p-nNOS-S847 and thus attenuate nNOS activation and simultaneously reduce the SNO-Src, p-Src, and NR2B tyrosine phosphorylation. Taken together, the S-nitrosylation of Src is caused by nNOS/NO signal, which is overactivated via Ca2+ influx dependent on NR2B/NMDAR, and subsequently facilitates Src auto-tyrosine phosphorylation and further phosphorylates NR2B. The "NR2B/NMDAR-nNOS/NO-SNO-Src-p-Src-NR2B/NMDAR" signaling cycle may be the molecular basis of NR2B tyrosine phosphorylation upward positive feedback, which demonstrates the possibility as one latent target for dyskinesia therapy.

Neuroprotective Mechanisms of Lycium barbarum Polysaccharides Against Ischemic Insults by Regulating NR2B and NR2A Containing NMDA Receptor Signaling Pathways

Glutamate excitotoxicity plays an important role in neuronal death after ischemia. However, all clinical trials using glutamate receptor inhibitors have failed. This may be related to the evidence that activation of different subunit of NMDA receptor will induce different effects. Many studies have shown that activation of the intrasynaptic NR2A subunit will stimulate survival signaling pathways, whereas upregulation of extrasynaptic NR2B will trigger apoptotic pathways. A Lycium barbarum polysaccharide (LBP) is a mixed compound extracted from Lycium barbarum fruit. Recent studies have shown that LBP protects neurons against ischemic injury by anti-oxidative effects. Here we first reported that the effect of LBP against ischemic injury can be achieved by regulating NR2B and NR2A signaling pathways. By in vivo study, we found LBP substantially reduced CA1 neurons from death after transient global ischemia and ameliorated memory deficit in ischemic rats. By in vitro study, we further confirmed that LBP increased the viability of primary cultured cortical neurons when exposed to oxygen-glucose deprivation (OGD) for 4 h. Importantly, we found that LBP antagonized increase in expression of major proteins in the NR2B signal pathway including NR2B, nNOS, Bcl-2-associated death promoter (BAD), cytochrome C (cytC) and cleaved caspase-3, and also reduced ROS level, calcium influx and mitochondrial permeability after 4 h OGD. In addition, LBP prevented the downregulation in the expression of NR2A, pAkt and pCREB, which are important cell survival pathway components. Furthermore, LBP attenuated the effects of a NR2B co-agonist and NR2A inhibitor on cell mortality under OGD conditions. Taken together, our results demonstrated that LBP is neuroprotective against ischemic injury by its dual roles in activation of NR2A and inhibition of NR2B signaling pathways, which suggests that LBP may be a superior therapeutic candidate for targeting glutamate excitotoxicity for the treatment of ischemic stroke.

Neuroprotective effects of vitexin against isoflurane-induced neurotoxicity by targeting the TRPV1 and NR2B signaling pathways

Vitexin is a bioactive compound extracted from hawthorn leaves, which reduces blood pressure and has anti‑inflammatory and potential anticancer effects. However, the mechanisms underlying the protective effects of vitexin against isoflurane‑induced neurotoxicity remain elusive. Therefore, the aim of the present study was to investigate these mechanisms further. Sprague Dawley rats received 1.4% isoflurane in a 100% oxygen environment for 2h. Human PC12 pheochromocytoma neurosecretory cells were exposed to 2% isoflurane for 12h before they were treated with 1, 10 or 100µM vitexin for a further 24h. Vitexin inhibited the isoflurane-induced cell cytotoxicity and weakened isoflurane-induced neuroinflammation and oxidative stress pathways in PC12 cells. In addition, treatment with vitexin suppressed isoflurane‑induced caspase‑3 activation and increased β-secretase1 levels in PC12 cells. Furthermore, vitexin treatment decreased the levels of isoflurane‑induced cytosolic calcium and reactive oxygen species, and downregulated the expression of transient receptor potential cation channel subfamily V member1 (TRPV1) and glutamate ionotropic receptor NMDA type subunit 2B (NR2B) protein expression in isoflurane-treated PC12 cells. These results suggest that vitexin mediates its protective effects against isoflurane-induced neurotoxicity by targeting the TRPV1 and NR2B signaling pathways.

Role of Fyn-mediated NMDA receptor function in prediabetic neuropathy in mice.

Diabetic neuropathy is a common complication of diabetes. This study evaluated the role of Fyn kinase and N-methyl-d-aspartate receptors (NMDARs) in the spinal cord in diabetic neuropathy using an animal model of high-fat diet-induced prediabetes. We found that prediabetic wild-type mice exhibited tactile allodynia and thermal hypoalgesia after a 16-wk high-fat diet, relative to normal diet-fed wild-type mice. Furthermore, prediabetic wild-type mice exhibited increased tactile allodynia and thermal hypoalgesia at 24 wk relative to 16 wk. Such phenomena were correlated with increased expression and activation of NR2B subunit of NMDARs, as well as Fyn-NR2B interaction in the spinal cord. Fyn(-/-) mice developed prediabetes after 16-wk high-fat diet treatment and exhibited thermal hypoalgesia, without showing tactile allodynia or altered expression and activation of NR2B subunit, relative to normal diet-fed Fyn(-/-) mice. Finally, intrathecal administrations of Ro 25-6981 (selective NR2B subunit-containing NMDAR antagonist) dose-dependently alleviated tactile allodynia, but not thermal hypoalgesia, at 16 and 24 wk in prediabetic wild-type mice. Our results suggested that Fyn-mediated NR2B signaling plays a critical role in regulation of prediabetic neuropathy and that the increased expression/function of NR2B subunit-containing NMDARs may contribute to the progression of neuropathy in type 2 diabetes.

Regulation of fear extinction versus other affective behaviors by discrete cortical scaffolding complexes associated with NR2B and PKA signaling.

In patients suffering from post-traumatic stress disorder (PTSD), fear evoked by trauma-related memories lasts long past the traumatic event and it is often complicated by general anxiety and depressed mood. This poses a treatment challenge, as drugs beneficial for some symptoms might exacerbate others. For example, in preclinical studies, antagonists of the NR2B subunit of N-methyl-d-aspartate receptors and activators of cAMP-dependent protein kinase (PKA) act as potent antidepressants and anxiolytics, but they block fear extinction. Using mice, we attempted to overcome this problem by interfering with individual NR2B and PKA signaling complexes organized by scaffolding proteins. We infused cell-permeable Tat peptides that displaced either NR2B from receptor for activated C kinase 1 (RACK1), or PKA from A-kinase anchor proteins (AKAPs) or microtubule-associated proteins (MAPs). The infusions were targeted to the retrosplenial cortex, an area involved in both fear extinction of remotely acquired memories and in mood regulation. Tat-RACK1 and Tat-AKAP enhanced fear extinction, all peptides reduced anxiety and none affected baseline depression-like behavior. However, disruption of PKA complexes distinctively interfered with the rapid antidepressant actions of the N-methyl-D-aspartate receptors antagonist MK-801 in that Tat-MAP2 blocked, whereas Tat-AKAP completely inverted the effect of MK-801 from antidepressant to depressant. These effects were unrelated to the MK-801-induced changes of brain-derived neurotrophic factor messenger RNA levels. Together, the findings suggest that NR2B–RACK1 complexes specifically contribute to fear extinction, and may provide a target for the treatment of PTSD. AKAP-PKA, on the other hand, appears to modulate fear extinction and antidepressant responses in opposite directions.

Mild Hypothermia Combined with Hydrogen Sulfide Treatment During Resuscitation Reduces Hippocampal Neuron Apoptosis Via NR2A, NR2B, and PI3K-Akt Signaling in a Rat Model of Cerebral Ischemia-Reperfusion Injury.

We investigated whether mild hypothermia combined with sodium hydrosulfide treatment during resuscitation improves neuron survival following cerebral ischemia-reperfusion injury beyond that observed for the individual treatments. Male Sprague-Dawley rats were divided into seven groups (n = 20 for each group). All rats underwent Pulsinelli 4-vessel occlusion. Ischemia was induced for 15min using ligatures around the common carotid arteries, except for the sham group. Immediately after initiating reperfusion, the mild hypothermia (MH), sodium hydrosulfide (NaHS), hydroxylamine (HA), MH + NaHS, MH + HA, and ischemia-reperfusion (I/R) control groups received an intraperitoneal injection of saline, sodium hydrosulfide, hydroxylamine, sodium hydrosulfide, hydroxylamine, and saline, respectively, and mild hypothermia (32 to 33°C) was induced in the MH, MH + NaHS, and MH + HA groups for 6h. The levels of NR2A, NR2B, p-Akt, and p-Gsk-3β in the hippocampus of the MH, NaHS, and MH + NaHS groups were higher than those in the I/R control group, with the highest levels observed in the MH + NaHS group (P < 0.05). Treatment with hydroxylamine reduced the levels of these proteins in the HA and MH + HA groups, compared with the I/R control and MH groups, respectively. The apoptotic index of the CA1 region of the hippocampus was 45.2, 66.5, 63.5, and 84.8% in the MH + NaHS, MH, NaHS, and I/R control groups, respectively (P < 0.05), indicating that the combination treatment shifted the NR2A/NR2B balance in favor of synaptic neuron stimulation and phosphatidylinositol 3'-kinase (PI3K)/Akt signaling. The combination of mild hypothermia and sodium hydrosulfide treatment for resuscitation following ischemia-reperfusion injury was more beneficial for reducing hippocampal apoptosis and pathology than that of mild hypothermia or hydrogen sulfide treatment alone.

Neuroprotective effect of estrogen: Role of nonsynaptic NR2B-containing NMDA receptors

Excessive activation of N-methyl-D-aspartate receptors (NMDARs) has been implicated in the pathophysiology of chronic neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and Huntington's disease. Some studies reported that NR2A and NR2B play different roles in the central nervous system (CNS). The NR2A subunit is primarily found in the synapses and is required for glutamate-mediated neuronal survival. On the other hand, the NR2B subunit is primarily found in the extrasynaptic sites and is required for glutamate-mediated neuronal death in both in vitro and in vivo experiments. Estrogen is a steroid hormone well known for its widespread effects such as neuroprotection in the brain. Classically, estrogen can bind to two kinds of nuclear receptors, namely, estrogen receptor α (ERα) and estrogen receptor β (ERβ), and produce physiological and neuroprotective effects. Aside from nuclear receptors, estrogen has one membrane receptor, which can either be G-protein-coupled receptor 30 (GPR30), Gq-mER, or ER-X. NMDA exposure clearly promotes NR2B subunit phosphorylation at Ser-1303 and causes neuronal cell death. GPR30 mediates rapid non-genomic effects to protect neurons against injury by inhibiting p-DAPK1 dephosphorylation, which inhibits NR2B subunit phosphorylation at Ser-1303. In addition, NMDA exposure and global ischemia activate the autophagy pathway and induce cell death, which are markedly blocked by the NR2B antagonist Ro 25-6981. Thus, NR2B signaling, autophagy induction and cell death may be closely related. Ro 25-6981 inhibits the dissociation of the NR2B-Beclin-1 signaling complex and delays autophagy in vivo, thus confirming the link between NR2B signaling and autophagy. In short, ERα, ERβ, and GPR30 are involved in the neuroprotection of estrogen in the CNS. Additional research must be conducted to reveal the mechanism of estrogen action fully and to identify better targets for the development of more effective drugs. This article is part of a Special Issue entitled 'Extrasynaptic ionotropic receptors'.

Spinal SIRPα1-SHP2 interaction regulates spinal nerve ligation-induced neuropathic pain via PSD-95-dependent NR2B activation in rats

The fact that neuropathic pain mechanisms are not well understood is a major impediment in the development of effective clinical treatments. We examined whether the interaction between signal regulatory protein alpha 1 (SIRPα1) and Src homology-2 domain-containing protein tyrosine phosphatase 2 (SHP2), and the downstream spinal SHP2/postsynaptic density 95 (PSD-95)/N-methyl-d-aspartate receptor NR2B subunit signaling cascade play a role in neuropathic pain. Following spinal nerve ligation (L5), we assessed tactile allodynia using the von Frey filament test and analyzed dorsal horn samples (L4-5) by Western blotting, reverse transcription polymerase chain reaction, coimmunoprecipitation, and immunofluorescence. Nerve ligation induced allodynia, SIRPα1, SHP2, phosphorylated SHP2 (pSHP2), and phosphorylated NR2B (pNR2B) expression, and SHP2-PSD-95, pSHP2-PSD-95, PSD-95-NR2B, and PSD-95-pNR2B coimmunoprecipitation in the ipsilateral dorsal horn. In allodynic rats, injury-induced SHP2 immunoreactivity was localized in the ipsilateral dorsal horn neurons and coincident with PSD-95 and NR2B immunoreactivity. SIRPα1 silencing using small interfering RNA (siRNA; 1, 3, or 5μg/rat for 7days) prevented injury-induced allodynia and the associated changes in protein expression, phosphorylation, and coimmunoprecipitation. Intrathecal administration of NSC-87877 (an SHP2 antagonist; 1, 10, or 100μM/rat) and SIRPα1-neutralizing antibodies (1, 10, or 30μg/rat) suppressed spinal nerve ligation-induced allodynia, spinal SHP2 and NR2B phosphorylation, and SHP2/phosphorylated SHP2-PSD-95 and PSD-95-NR2B/phosphorylated NR2B coprecipitation. SHP2 siRNA led to similar effects as the NSC-87877 and SIRPα1 antibody treatments, except it prevented the allodynia-associated spinal SHP2 expression. In conclusion, our results suggest that a spinal SIRPα1-SHP2 interaction exists that subsequently triggers SHP2/PSD-95/NR2B signaling, thereby playing a role in neuropathic pain development.

Distinct Modes of AMPA Receptor Suppression at Developing Synapses by GluN2A and GluN2B: Single-Cell NMDA Receptor Subunit Deletion In Vivo

During development there is an activity-dependent switch in synaptic N-Methyl-D-aspartate (NMDA) receptor subunit composition from predominantly GluN2B to GluN2A, though the precise role of thisswitch remains unknown. By deleting GluN2 subunits in single neurons during synaptogenesis, we find that both GluN2B and GluN2A suppress AMPA receptor expression, albeit by distinct means. Similar to GluN1, GluN2B deletion increases the number of functional synapses, while GluN2A deletion increases the strength of unitary connections without affecting the number of functional synapses. We propose a model of excitatory synapse maturation in which baseline activation of GluN2B-containing receptors prevents premature synapse maturation until correlated activity allows induction of functional synapses. This activity also triggers the switch to GluN2A, which dampens further potentiation. Furthermore, we analyze the subunit composition of synaptic NMDA receptors in CA1 pyramidal cells, provide electrophysiological evidence fora large population of synaptic triheteromeric receptors, and estimate the subunit-dependent open probability.

Properties of glutamatergic synapses in immature layer Vb pyramidal neurons: coupling of pre- and postsynaptic maturational states

Following initial contact formation, glutamatergic synapses in cortical neurons undergo pronounced functional maturation. These maturational events, occurring both pre- and postsynaptically, have been well described in the developing hippocampus. In this paper, we characterized glutamatergic synapses in immature layer Vb pyramidal neurons of the mouse somatosensory cortex during early postnatal development. At postnatal day 7, a significant subpopulation of glutamatergic synapses exhibited a low release probability that was accompanied by strong paired-pulse facilitation of AMPA EPSCs (paired-pulse ratio C > or = 2). Increasing extracellular Ca(2+) concentration increased release probability and led to paired-pulse depression. During further postnatal development, these functionally immature synapses disappeared. As shown pharmacologically,these synapses expressed postsynaptic NMDA receptors containing NR2B subunits, while NMDA receptors with NR2A subunits were lacking. Taken together, a low release probability presynaptically was coupled to postsynaptic NR2B signaling. This subpopulation of neocortical synapses thus differed from the majority of synapses in the developing hippocampus, where high release probability is coupled to NR2B signaling. The novel type of functionally immature glutamatergic synapse described here might play an important role in early developmental synapse elimination and in the activity-dependent refinement of the neocortical synaptic microcircuitry.

A novel protein complex in membrane rafts linking the NR2B glutamate receptor and autophagy is disrupted following traumatic brain injury.

Hyperactivation of N-methyl-D-aspartate receptors (NRs) is associated with neuronal cell death induced by traumatic brain injury (TBI) and many neurodegenerative conditions. NR signaling efficiency is dependent on receptor localization in membrane raft microdomains. Recently, excitotoxicity has been linked to autophagy, but mechanisms governing signal transduction remain unclear. Here we have identified protein interactions between NR2B signaling intermediates and the autophagic protein Beclin-1 in membrane rafts of the normal rat cerebral cortex. Moderate TBI induced rapid recruitment and association of NR2B and pCaMKII to membrane rafts, and translocation of Beclin-1 out of membrane microdomains. Furthermore, TBI caused significant increases in expression of key autophagic proteins and morphological hallmarks of autophagy that were significantly attenuated by treatment with the NR2B antagonist Ro 25-6981. Thus, stimulation of autophagy by NR2B signaling may be regulated by redistribution of Beclin-1 in membrane rafts after TBI.

NR2B Signaling Regulates the Development of Synaptic AMPA Receptor Current

The postnatal maturation of glutamatergic synapses involves a change in composition and functional contribution of postsynaptic receptors. Developing cortical synapses are dominated by NMDA receptors (NMDARs) containing NR2B subunits and are characterized by a low ratio of AMPA/NMDA receptor-mediated current. Synapse maturation is marked by the incorporation of NR2A-containing NMDA receptors and an increase in the AMPA/NMDA current ratio. We show here that NMDARs containing the NR2B subunit regulate glutamatergic transmission at developing synapses by negatively influencing the synaptic incorporation of AMPA receptors (AMPARs). Genetic removal of NR2B leads to increased surface expression and synaptic localization of AMPA receptor subunits and a corresponding increase in AMPAR-mediated synaptic current. Enrichment of synaptic AMPARs, in the absence of NR2B signaling, is associated with increased levels of transmembrane AMPAR regulatory protein (TARP) expression and is blocked by expression of a dominant-negative TARP construct (gamma-2deltaC). These observations suggest that NR2B signaling limits AMPA receptor incorporation at developing synapses by negatively regulating TARP expression and provide a mechanism to explain the maintenance of low AMPA/NMDA ratio at immature glutamatergic synapses.

Odorant Deprivation Reversibly Modulates Transsynaptic Changes in the NR2B-Mediated CREB Pathway in Mouse Piriform Cortex

The olfactory system is an outstanding model for understanding activity-dependent neuronal plasticity in mammals. Olfactory sensory neurons (OSNs) in the periphery project onto mitral/tufted cells in the olfactory bulb (OB) and these mitral/tufted cells in turn project to piriform cortex (PC). Numerous studies have examined changes in OB after a permanent OSN ablation, but little is known about "trans-transsynaptic" changes taking place in the PC. Permanent zinc sulfate lesion of the olfactory epithelium resulted in a selective loss of the NMDA receptor NR2B protein and mRNA expression in pyramidal cells in layer IIb of PC after 2-7 d. Regulatory elements affected by NR2B signaling, namely the phosphorylation of CREB, were also downregulated only in layer IIb neurons. These changes could be caused by OSN axon loss in the zinc sulfate lesion, or to a reduced activity. To test this hypothesis, we performed both permanent and reversible naris occlusion, which blocks odorant access to the nasal cavities and OSN activity. The expression of NR2B and phospho-CREB were downregulated 5 d after occlusion and this reduction was fully restored 10 d after reopening of the naris. Subsequently, we identified the subset of pyramidal cells in layer IIb that are especially sensitive to the loss of odor-evoked activity using double retrograde tracers. In summary, the present study provides an initial characterization of the molecular mechanisms associated with odor stimulation on second order neuronal plasticity and phenotype in the olfactory system.

Anti-inflammatory drugs: What is safe?

The olfactory system is an outstanding model for understanding activity-dependent neuronal plasticity in mammals. Olfactory sensory neurons (OSNs) in the periphery project onto mitral/tufted cells in the olfactory bulb (OB) and these mitral/tufted cells in turn project to piriform cortex (PC). Numerous studies have examined changes in OB after a permanent OSN ablation, but little is known about “trans-transsynaptic” changes taking place in the PC. Permanent zinc sulfate lesion of the olfactory epithelium resulted in a selective loss of the NMDA receptor NR2B protein and mRNA expression in pyramidal cells in layer IIb of PC after 2–7 d. Regulatory elements affected by NR2B signaling, namely the phosphorylation of CREB, were also downregulated only in layer IIb neurons. These changes could be caused by OSN axon loss in the zinc sulfate lesion, or to a reduced activity. To test this hypothesis, we performed both permanent and reversible naris occlusion, which blocks odorant access to the nasal cavities and OSN activity. The expression of NR2B and phospho-CREB were downregulated 5 d after occlusion and this reduction was fully restored 10 d after reopening of the naris. Subsequently, we identified the subset of pyramidal cells in layer IIb that are especially sensitive to the loss of odor-evoked activity using double retrograde tracers. In summary, the present study provides an initial characterization of the molecular mechanisms associated with odor stimulation on second order neuronal plasticity and phenotype in the olfactory system.

Brain-derived neurotrophic factor enhances association of protein tyrosine phosphatase PTP1D with the NMDA receptor subunit NR2B in the cortical postsynaptic density

Our recent studies revealed that brain-derived neurotrophic factor (BDNF) rapidly enhances tyrosine phosphorylation and dephosphorylation of the NMDA receptor subunit, NR2B, in the postsynaptic density (PSD), potentially regulating synaptic plasticity. To explore the molecular mechanisms underlying synaptic NR2B signaling, we examined the protein tyrosine phosphatase, PTP1D; BDNF reportedly increases association of PTP1D with tyrosine phosphorylated proteins in cortical neurons and PC 12 cells. We now report that PTP1D is an intrinsic component of the rat cerebrocortical PSD, based on Western blot analysis using specific anti-PTP1D antibodies. In addition, NR2B was co-immunoprecipitated with PTP1D using anti-NR2B antibodies or anti-PTP1D antibodies, indicating physical association of the subunit with PTP1D. Moreover, treatment of the purified PSD with BDNF for 5 min elicited a two-fold increase in the association of NR2B with PTP1D. The BDNF action appeared to be specific, since nerve growth factor, another member of the neurotrophin gene family, did not alter the association. Finally, an overlay assay revealed that BDNF caused a two-fold increase in binding of blotted PSD NR2B proteins to PTP1D-SH2 domains, revealing molecular mechanisms mediating the PTP1D-NR2B binding. Taken together, our results raise the possibility that PTP1D participates in BDNF-mediated NR2B signaling cascades at the postsynaptic site, thereby regulating synaptic plasticity.