Nefiracetam activation of CaM kinase II and protein kinase C mediated by NMDA and metabotropic glutamate receptors in olfactory bulbectomized mice

Aberrant behaviors related to learning and memory in olfactory bulbectomized (OBX) mice have been documented in the previous studies. We reported that the impairment of long-term potentiation (LTP) of hippocampal CA1 regions from OBX mice was associated with down-regulation of CaM kinase II (CaMKII) and protein kinase C (PKC) activities. We now demonstrated that the nootropic drug, nefiracetam, significantly improved spatial reference memory-related behaviors as assessed by Y-maze and novel object recognition task in OBX mice. Nefiracetam also restored hippocampal LTP injured in OBX mice. Nefiracetam treatment restored LTP-induced PKCα (Ser657) and NR1 (Ser896) phosphorylation as well as increase in their basal phosphorylation in the hippocampal CA1 region of OBX mice. Likewise, nefiracetam improved LTP-induced CaMKIIα (Thr286) autophosphorylation and GluR1 (Ser831) phosphorylation and increased their basal phosphorylation. The enhancement of PKCα (Ser657) and CaMKIIα (Thr286) autophosphorylation by nefiracetam was inhibited by treatment with (±)-α-Methyl-(4-carboxyphenyl)glycine and DL-2-Amino-5-phosphonovaleric acid, respectively. The enhancement of LTP induced by nefiracetam is inhibited by treatment with 2-methyl-6-(phenylethynyl)-pyridine, but not by treatment with LY367385, suggesting that metabotropic glutamate receptor 5 (mGluR5) but not mGluR1 is involved in the nefiracetam-induced LTP enhancement. Taken together, nefiracetam ameliorates OBX-induced deficits in memory-related behaviors and impairment of LTP in the hippocampal CA1 region through activation of NMDAR and mGluR5, thereby leading to an increase in activities of CaMKIIα (Thr286) and PKCα (Ser657), respectively.     

Decreased calcium/calmodulin-dependent protein kinase II and protein kinase C activities mediate impairment of hippocampal long-term potentiation in the olfactory bulbectomized mice

Olfactory bulbectomized (OBX) mice showed significant impairment of learning and memory-related behaviors 14 days after olfactory bulbectomy, as measured by passive avoidance and Y-maze tasks. We here observed a large impairment of hippocampal long-term potentiation (LTP) in the OBX mice. Concomitant with decreased acetylcholinesterase expression, protein kinase C (PKC)alpha autophosphorylation and NR1(Ser-896) phosphorylation significantly decreased in the hippocampal CA1 region of OBX mice. Both PKCalpha and NR1(Ser-896) phosphorylation significantly increased following LTP in the control mice, whereas increases were not observed in OBX mice. Like PKC activities, calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation significantly decreased in the hippocampal CA1 region of OBX mice as compared with that of control mice. In addition, increased CaMKII autophosphorylation following LTP was not observed in OBX mice. Finally, the impairment of CaMKII autophosphorylation was closely associated with reduced pGluR1(Ser-831) phosphorylation, without change in synapsin I (site 3) phosphorylation in the hippocampal CA1 region of OBX mice. Taken together, in OBX mice NMDA receptor hypofunction, possibly through decreased PKCalpha activity, underlies decreased CaMKII activity in the post-synaptic regions, thereby impairing LTP induction in the hippocampal CA1 region. Both decreased PKC and CaMKII activities with concomitant LTP impairment account for the learning disability observed in OBX mice.     

Protein kinase C promotes N-methyl-D-aspartate (NMDA) receptor trafficking by indirectly triggering calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation

Regulation of neuronal NMDA receptor (NMDAR) is critical in synaptic transmission and plasticity. Protein kinase C (PKC) promotes NMDAR trafficking to the cell surface via interaction with NMDAR-associated proteins (NAPs). Little is known, however, about the NAPs that are critical to PKC-induced NMDAR trafficking. Here, we showed that calcium/calmodulin-dependent protein kinase II (CaMKII) could be a NAP that mediates the potentiation of NMDAR trafficking by PKC. PKC activation promoted the level of autophosphorylated CaMKII and increased association with NMDARs, accompanied by functional NMDAR insertion, at postsynaptic sites. This potentiation, along with PKC-induced long term potentiation of the AMPA receptor-mediated response, was abolished by CaMKII antagonist or by disturbing the interaction between CaMKII and NR2A or NR2B. Further mutual occlusion experiments demonstrated that PKC and CaMKII share a common signaling pathway in the potentiation of NMDAR trafficking and long-term potentiation (LTP) induction. Our results revealed that PKC promotes NMDA receptor trafficking and induces synaptic plasticity through indirectly triggering CaMKII autophosphorylation and subsequent increased association with NMDARs.     

Activation of D1 dopamine receptors increases surface expression of AMPA receptors and facilitates their synaptic incorporation in cultured hippocampal neurons

Considerable evidence indicates that neuroadaptations leading to addiction involve the same cellular processes that enable learning and memory, such as long-term potentiation (LTP), and that psychostimulants influence LTP through dopamine (DA)-dependent mechanisms. In hippocampal CA1 pyramidal neurons, LTP involves insertion of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors into excitatory synapses. We used dissociated cultures to test the hypothesis that D1 family DA receptors influence synaptic plasticity in hippocampal neurons by modulating AMPA receptor trafficking. Brief exposure (5 min) to a D1 agonist increased surface expression of glutamate receptor (GluR)1-containing AMPA receptors by increasing their rate of externalization at extrasynaptic sites. This required the secretory pathway but not protein synthesis, and was mediated mainly by protein kinase A (PKA) with a smaller contribution from Ca2+-calmodulin-dependent protein kinase II (CaMKII). Prior D1 receptor stimulation facilitated synaptic insertion of GluR1 in response to subsequent stimulation of synaptic NMDA receptors with glycine. Our results support a model for synaptic GluR1 incorporation in which PKA is required for initial insertion into the extrasynaptic membrane whereas CaMKII mediates translocation into the synapse. By increasing the size of the extrasynaptic GluR1 pool, D1 receptors may promote LTP. Psychostimulants may usurp this mechanism, leading to inappropriate plasticity that contributes to addiction-related behaviors.     

Unique mechanism of action of Alzheimer's drugs on brain nicotinic acetylcholine receptors and NMDA receptors

While a variety of hypotheses have been proposed for the cause of Alzheimer's disease, our knowledge is far from complete to explain the disease making it difficult to develop the methods for treatment. In the brain of Alzheimer's patients, both neuronal nicotinic acetylcholine (nACh) receptors and NMDA receptors are known to be down-regulated. Thus four anticholinesterases have been developed and approved for the treatment in the U.S.A. However, these are not ideal drugs considering their side effects and limited effectiveness. Nefiracetam is being developed for the treatment of Alzheimer's and other patients with dementia, and has unique actions in potentiating the activity of both nACh and NMDA receptors as demonstrated by in vitro patch clamp experiments using rat cortical neurons in primary culture. Nefiracetam potentiated alpha4beta2-like ACh- and NMDA-induced currents at nanomolar concentrations forming bell-shaped dose-response curves with the maximum potentiation occurring at 1 and 10 nM, respectively. Nefiracetam potentiated nACh receptor currents via G(s) proteins, but not G(i)/G(o) proteins, PKA or PKC. Nefiracetam potentiation of NMDA currents occurred via interactions with the glycine binding site of the NMDA receptor. The nefiracetam potentiation of both nACh and NMDA receptors in a potent and efficacious manner is deemed responsible for its cognitive enhancing action.     

Long-term potentiation in cultured hippocampal neurons

Studies performed on low-density primary neuronal cultures have enabled dissection of molecular and cellular changes during N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP). Various electrophysiological and chemical induction protocols were developed for the persistent enhancement of excitatory synaptic transmission in hippocampal neuronal cultures. The characterisation of these plasticity models confirmed that they share many key properties with the LTP of CA1 neurons, extensively studied in hippocampal slices using electrophysiological techniques. For example, LTP in dissociated hippocampal neuronal cultures is also dependent on Ca(2+) influx through post-synaptic NMDA receptors, subsequent activation and autophosphorylation of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and an increase in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor insertion at the post-synaptic membrane. The availability of models of LTP in cultured hippocampal neurons significantly facilitated the monitoring of changes in endogenous postsynaptic receptor proteins and the investigation of the associated signalling mechanisms that underlie LTP. A central feature of LTP of excitatory synapses is the recruitment of AMPA receptors at the postsynaptic site. Results from the use of cell culture-based models started to establish the mechanism by which synaptic input controls a neuron's ability to modify its synapses in LTP. This review focuses on key features of various LTP induction protocols in dissociated hippocampal neuronal cultures and the applications of these plasticity models for the investigation of activity-induced changes in native AMPA receptors.     

Phosphorylation of proteins involved in activity-dependent forms of synaptic plasticity is altered in hippocampal slices maintained in vitro

The acute hippocampal slice preparation has been widely used to study the cellular mechanisms underlying activity-dependent forms of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD). Although protein phosphorylation has a key role in LTP and LTD, little is known about how protein phosphorylation might be altered in hippocampal slices maintained in vitro. To begin to address this issue, we examined the effects of slicing and in vitro maintenance on phosphorylation of six proteins involved in LTP and/or LTD. We found that AMPA receptor (AMPAR) glutamate receptor 1 (GluR1) subunits are persistently dephosphorylated in slices maintained in vitro for up to 8 h. alpha calcium/calmodulin-dependent kinase II (alphaCamKII) was also strongly dephosphorylated during the first 3 h in vitro but thereafter recovered to near control levels. In contrast, phosphorylation of the extracellular signal-regulated kinase ERK2, the ERK kinase MEK, proline-rich tyrosine kinase 2 (Pyk2), and Src family kinases was significantly, but transiently, increased. Electrophysiological experiments revealed that the induction of LTD by low-frequency synaptic stimulation was sensitive to time in vitro. These findings indicate that phosphorylation of proteins involved in N-methyl-D-aspartate (NMDA) receptor-dependent forms of synaptic plasticity is altered in hippocampal slices and suggest that some of these changes can significantly influence the induction of LTD.     

Sigma-1 receptor stimulation by dehydroepiandrosterone ameliorates cognitive impairment through activation of CaM kinase II, protein kinase C and extracellular signal-regulated kinase in olfactory bulbectomized mice

Dehydroepiandrosterone (DHEA) is one of the most abundant neurosteroids synthesized de novo in the CNS. We here found that sigma-1 receptor stimulation by DHEA improves cognitive function through phosphorylation of synaptic proteins in olfactory bulbectomized (OBX) mouse hippocampus. We have previously reported that calcium/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) were impaired in OBX mouse hippocampus. OBX mice were administered once a day for 7-8 days with DHEA (30 or 60 mg/kg p.o.) 10 days after operation. The spatial, cognitive and conditioned fear memories in OBX mice were significantly improved as assessed by Y-maze, novel object recognition and passive avoidance task, respectively. DHEA also improved impaired hippocampal long-term potentiation in OBX mice. Notably, DHEA treatment restored PKCα (Ser-657) autophosphorylation and NR1 (Ser-896) and myristoylated alanine-rich protein kinase C substrate (Ser-152/156) phosphorylation to the control levels in the hippocampal CA1 region. Likewise, DHEA treatment improved CaMKIIα (Thr-286) autophosphorylation and GluR1 (Ser-831) phosphorylation to the control levels in the CA1 region. Furthermore, DHEA treatment improved ERK and cAMP-responsive element-binding protein (Ser-133) phosphorylation to the control levels. Finally, NE-100, sigma-1 receptor antagonist, significantly inhibited the DHEA-induced improvement of memory-related behaviors and CaMKII, PKC and ERK phosphorylation in CA1 region. Taken together, sigma-1 receptor stimulation by DHEA ameliorates OBX-induced impairment in memory-related behaviors and long-term potentiation in the hippocampal CA1 region through activation of CaMKII, PKC and ERK.     

Facilitation of AMPA receptor synaptic delivery as a molecular mechanism for cognitive enhancement

Cell adhesion molecules and downstream growth factor-dependent signaling are critical for brain development and synaptic plasticity, and they have been linked to cognitive function in adult animals. We have previously developed a mimetic peptide (FGL) from the neural cell adhesion molecule (NCAM) that enhances spatial learning and memory in rats. We have now investigated the cellular and molecular basis of this cognitive enhancement, using biochemical, morphological, electrophysiological, and behavioral analyses. We have found that FGL triggers a long-lasting enhancement of synaptic transmission in hippocampal CA1 neurons. This effect is mediated by a facilitated synaptic delivery of AMPA receptors, which is accompanied by enhanced NMDA receptor-dependent long-term potentiation (LTP). Both LTP and cognitive enhancement are mediated by an initial PKC activation, which is followed by persistent CaMKII activation. These results provide a mechanistic link between facilitation of AMPA receptor synaptic delivery and improved hippocampal-dependent learning, induced by a pharmacological cognitive enhancer.     

Potassium Chloride Pulse Enhances Mitogen-Activated Protein Kinase Activity in Rat Hippocampal Slices

Abstract: Mitogen-activated protein (MAP) kinases have been implicated in multiple responses to extracellular stimuli. In this study we show that MAP kinase activity is enhanced after a KCI pulse. This activation correlates with an increased tyrosine phosphorylation of a 42-kDa protein as determined by antiphosphotyrosine immunoblot. The same band is found in an anti-MAP kinase immunoblot. Activity is enhanced within 1 min, reaches a maximum at 2 min, and returns to basal level after 10 min. A second peak of activity is observed between 12 and 30 min. The activation is completely blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), showing the involvement of the AMPA type of glutamate receptor. Partial inhibition of MAP kinase activation by 2-amino-5-phosphonovalerate (APV) also shows the involvement of the NMDA receptor. Because the KCI pulse used induces long-term potentiation (LTP) in rat hippocampal slice, we conclude that MAP kinase may be involved in neuronal transduction events leading to LTP.     

The T-type voltage-gated calcium channel as a molecular target of the novel cognitive enhancer ST101: enhancement of long-term potentiation and CaMKII autophosphorylation in rat cortical slices

In this study, we report that spiro[imidazo[1,2-a]pyridine-3,2-indan]-2(3H)-one (ST101; previously coded as ZSET1446) targets T-type voltage-gated calcium channels in mediating improved cognition in the CNS. We prepared rat somatosensory cortical and hippocampal slices, treated them with 0.01 to 100 nM ST101, and performed immunoblotting and electrophysiological analyses using various voltage-gated calcium channel (VGCC) inhibitors. Treatment of rat cortical slices with a range of ST101 concentrations significantly increased calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation following a bell-shaped dose-response curve, with 0.1 nM ST101 representing the maximally effective concentration. protein kinase Cα autophosphorylation was also significantly increased by 0.1 nM ST101 treatment. ST101 treatment had a moderate effect on CaMKII autophosphorylation but no effect on hippocampal protein kinase Cα autophosphorylation in slice preparations. Consistent with increased cortical CaMKII autophosphorylation, AMPA-type glutamate receptor subunit 1 (Ser-831) phosphorylation as a CaMKII post-synaptic substrate was significantly increased by treatment with 0.1-1 nM ST101, whereas phosphorylation of the pre-synaptic substrate synapsin I (Ser-603) remained unchanged. Notably, enhanced CaMKII autophosphorylation seen following 0.1 nM ST101 treatment was significantly inhibited by pre-treatment with 1 μM mibefradil, a T-type VGCC inhibitor, but not with N-type (ω-conotoxin), P/Q-type (ω-agatoxin) or L-type (nifedipine) VGCC inhibitors. Similarly, 0.1 nM ST101 significantly potentiated long-term potentiation in cortical but not hippocampal slices. Enhanced long-term potentiation in cortical slices was totally inhibited by 1 μM mibefadil treatment. Finally, whole-cell patch-clamp analysis of Neuro2A cells over-expressing recombinant human Ca(V) 3.1 (α1G) T-channels and treated with 0.1 nM ST101 showed significant increases in T-type VGCC currents. These results indicate that T-type VGCCs are direct molecular targets for the novel cognitive enhancer ST101, a potential Alzheimer disease therapeutic.     

Characterization of the mechanism underlying the reversal of long term potentiation by low frequency stimulation at hippocampal CA1 synapses.

Reversal of long term potentiation (LTP) may function to increase the flexibility and storage capacity of neuronal circuits; however, the underlying mechanisms remain incompletely understood. We show that depotentiation induced by low frequency stimulation (LFS) (2 Hz, 10 min, 1200 pulses) was input-specific and dependent on N-methyl-d-aspartate (NMDA) receptor activation. The ability of LFS to reverse LTP was mimicked by a brief application of NMDA. This NMDA-induced depotentiation was blocked by adenosine A(1) receptor antagonist. However, the reversal of LTP by LFS was unaffected by metabotropic glutamate receptor antagonism. This LFS-induced depotentiation was specifically prevented by protein phosphatase (PP)1 inhibitors, okadaic acid, and calyculin A but not by the PP2A or PP2B inhibitors. Furthermore, by using phosphorylation site-specific antibodies, we found that LFS-induced depotentiation is associated with a persistent dephosphorylation of the GluR1 subunit of amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor at serine 831, a protein kinase C and calcium/calmodulin-dependent protein kinase II (CaMKII) substrate, but not at serine 845, a substrate of cAMP-dependent protein kinase. This effect was mimicked by bath-applied adenosine or NMDA and was specifically prevented by okadaic acid. Also, the increased phosphorylation of CaMKII at threonine 286 and the decreased PP activity seen with LTP were overcome by LFS, adenosine, or NMDA application. These results suggest that LFS erases LTP through an NMDA receptor-mediated activation of PP1 to dephosphorylate amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and CaMKII in the CA1 region of the hippocampus.     

Bidirectional synaptic plasticity regulated by phosphorylation of stargazin-like TARPs

Synaptic plasticity involves protein phosphorylation cascades that alter the density of AMPA-type glutamate receptors at excitatory synapses; however, the crucial phosphorylated substrates remain uncertain. Here, we show that the AMPA receptor-associated protein stargazin is quantitatively phosphorylated and that stargazin phosphorylation promotes synaptic trafficking of AMPA receptors. Synaptic NMDA receptor activity can induce both stargazin phosphorylation, via activation of CaMKII and PKC, and stargazin dephosphorylation, by activation of PP1 downstream of PP2B. At hippocampal synapses, long-term potentiation and long-term depression require stargazin phosphorylation and dephosphorylation, respectively. These results establish stargazin as a critical substrate in the bidirectional control of synaptic strength, which is thought to underlie aspects of learning and memory.     

Inhibiting Src family tyrosine kinase activity blocks glutamate signalling to ERK1/2 and Akt/PKB but not JNK in cultured striatal neurones

Glutamate receptor activation of mitogen-activated protein (MAP) kinase signalling cascades has been implicated in diverse neuronal functions such as synaptic plasticity, development and excitotoxicity. We have previously shown that Ca2+-influx through NMDA receptors in cultured striatal neurones mediates the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt/protein kinase B (PKB) through a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathway. Exposing neurones to the Src family tyrosine kinase inhibitor PP2, but not the inactive analogue PP3, inhibited NMDA receptor-induced phosphorylation of ERK1/2 and Akt/PKB in a concentration-dependent manner, and reduced cAMP response element-binding protein (CREB) phosphorylation. To establish a link between Src family tyrosine kinase-mediated phosphorylation and PI 3-kinase signalling, affinity precipitation experiments were performed with the SH2 domains of the PI 3-kinase regulatory subunit p85. This revealed a Src-dependent phosphorylation of a focal adhesion kinase (FAK)-p85 complex on glutamate stimulation. Demonstrating that PI3-kinase is not ubiquitously involved in NMDA receptor signal transduction, the PI 3-kinase inhibitors wortmannin and LY294002 did not prevent NMDA receptor Ca2+-dependent phosphorylation of c-Jun N-terminal kinase 1/2 (JNK1/2). Further, inhibiting Src family kinases increased NMDA receptor-dependent JNK1/2 phosphorylation, suggesting that Src family kinase-dependent cascades may physiologically limit signalling to JNK. These results demonstrate that Src family tyrosine kinases and PI3-kinase are pivotal regulators of NMDA receptor signalling to ERK/Akt and JNK in striatal neurones.     

Expression mechanisms underlying long-term potentiation: a postsynaptic view

This review summarizes the various experiments that have been carried out to determine if the expression of long-term potentiation (LTP), in particular N-methyl-D-aspartate (NMDA) receptor-dependent LTP, is presynaptic or postsynaptic. Evidence for a presynaptic expression mechanism comes primarily from experiments reporting that glutamate overflow is increased during LTP and from experiments showing that the failure rate decreases during LTP. However, other experimental approaches, such as monitoring synaptic glutamate release by recording astrocytic glutamate transporter currents, have failed to detect any change in glutamate release during LTP. In addition, the discovery of silent synapses, in which LTP rapidly switches on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor function at NMDA-receptor-only synapses, provides a postsynaptic mechanism for the decrease in failures during LTP. It is argued that the preponderance of evidence favours a postsynaptic expression mechanism, whereby NMDA receptor activation results in the rapid recruitment of AMPA receptors as well as a covalent modification of synaptic AMPA receptors.     

CAKbeta/Pyk2 kinase is a signaling link for induction of long-term potentiation in CA1 hippocampus

Long-term potentiation (LTP) is an activity-dependent enhancement of synaptic efficacy, considered a model of learning and memory. The biochemical cascade producing LTP requires activation of Src, which upregulates the function of NMDA receptors (NMDARs), but how Src becomes activated is unknown. Here, we show that the focal adhesion kinase CAKbeta/Pyk2 upregulated NMDAR function by activating Src in CA1 hippocampal neurons. Induction of LTP was prevented by blocking CAKbeta/Pyk2, and administering CAKbeta/Pyk2 intracellularly mimicked and occluded LTP. Tyrosine phosphorylation of CAKbeta/Pyk2 and its association with Src was increased by stimulation that produced LTP. Finally, CAKbeta/Pyk2-stimulated enhancement of synaptic AMPA responses was prevented by blocking NMDARS, chelating intracellular Ca(2+), or blocking Src. Thus, activating CAKbeta/Pyk2 is required for inducing LTP and may depend upon downstream activation of Src to upregulate NMDA receptors.     

磷酸化钙/钙调蛋白依赖性蛋白激酶Ⅱ在大鼠脊髓背角C-纤维诱发电位长时程增强的诱导和维持中的作用

实验旨在探讨钙/钙调蛋白依赖性蛋白激酶II(calcium／calmodulin-dependent protein kinase Ⅱ,CaMKⅡ)在脊髓背角C-纤维诱发电位长时程增强(long—term potentiation,LTP)的诱导和维持中的作用。用Western blot技术分别检测LTP形成30min和3h脊髓背角(L4-L6)CaMKⅡ的含量及其磷酸化水平。同时观察脊髓局部给予CAMKⅡ选择性抑制剂KN-93后对脊髓背角LTP和CaMKII磷酸化的影响。观察结果如下：(1)诱导LTP后30min,CaMK Ⅱ的磷酸化水平明显高于对照组,而CaMKⅡ的总量无变化;诱导LTP后3h CaMKⅡ的磷酸化水平进一步升高。而且CaMKⅡ的总量也明显增加(n=4);(2)强直刺激前30min于脊髓局部给予CaMKⅡ的特异性抑制剂KN-93(100μmol/L),可阻断LTP的诱导,同时明显抑制CaMKⅡ的磷酸化水平;(3)诱导LTP后30min给予KN-93,可显著抑制LTP的维持,同时CaMKⅡ的磷酸化水平与未用药组相比也明显降低（n=3);(4)LTP3h后给予KN-93,LTP的幅值不受影响,磷酸化的CaMKⅡ的含量与用药前相比也无差别（n=3)。根据上述实验结果可以认为,CaMKⅡ的激活参与脊髓背角C-纤维诱发电位LTP的诱导和早期维持过程。     

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.     

NMDA receptor activation results in PKA- and ERK-dependent Mnk1 activation and increased eIF4E phosphorylation in hippocampal area CA1

Protein synthesis is essential for the stabilization of glutamate receptor-dependent forms of long-lasting hippocampal synaptic plasticity and for the consolidation of memory, but the signal transduction mechanisms that regulate translation factors during these processes are not well understood. As a first step towards understanding how translation is activated during synaptic plasticity, we investigated how the eukaryotic initiation factor 4E (eIF4E), a rate-limiting mRNA cap-binding protein, and its kinase, Mnk1, are regulated by protein kinase C (PKC), cAMP-dependent protein kinase (PKA) and N-methyl-D-aspartate (NMDA) receptor activation in hippocampal area CA1. We found that treatment of mouse hippocampal slices with either phorbol ester, to activate PKC, or forskolin, to activate PKA, resulted in activation of Mnk1 and increased eIF4E phosphorylation that was dependent on extracellular signal-regulated kinase (ERK). Similarly, brief treatment of hippocampal slices with NMDA resulted in activation of Mnk1 and increased phosphorylation of eIF4E. The NMDA-induced activation of Mnk1 and increased phosphorylation of eIF4E were dependent on PKA and ERK, but not PKC, and were present in synaptoneurosome preparations. Immunohistochemical analysis revealed that the PKA- and ERK-dependent increases in Mnk1 activation induced by NMDA also occurred in dendrites. These findings identify a specific regulatory pathway that can couple NMDA receptor activation to translation initiation factors in the hippocampus, and may represent a mechanism for triggering dendritic protein synthesis during long-term potentiation and long-term memory formation.     

突触长时程增强形成机制的研究进展

高等动物脑内突触传递的可塑性是近30年来神经科学研究的热点,突触传递长时程增强（long-term potentiation,LTP)是神经元可塑性的反映,其形成主要与突触后机制有关。过去关于LTP机制的研究主要集中于N－甲基－D门冬氨酸（NMDA）受体的特征及该受体被激活后的细胞内级联反应,现认为脑内存在只具有NMDA受体而不具有α－氨基羟甲基恶唑丙酸（AMPA）受体的“静寂突触（silent synapse)”,这一概念的提出,使人们认识到AMPA受体在LTP表达的突触后机制中的重要作用。