Novel nootropic drug sunifiram enhances hippocampal synaptic efficacy via glycine‐binding site of N‐methyl‐D‐aspartate receptor

Sunifiram is a novel pyrrolidone nootropic drug structurally related to piracetam, which was developed for neurodegenerative disorder like Alzheimer's disease. Sunifiram is known to enhance cognitive function in some behavioral experiments such as Morris water maze task. To address question whether sunifiram affects N‐methyl‐D ‐aspartate receptor (NMDAR)‐dependent synaptic function in the hippocampal CA1 region, we assessed the effects of sunifiram on NMDAR‐dependent long‐term potentiation (LTP) by electrophysiology and on phosphorylation of synaptic proteins by immunoblotting analysis. In mouse hippocampal slices, sunifiram at 10–100 nM significantly enhanced LTP in a bell‐shaped dose‐response relationship which peaked at 10 nM. The enhancement of LTP by sunifiram treatment was inhibited by 7‐chloro‐kynurenic acid (7‐ClKN), an antagonist for glycine‐binding site of NMDAR, but not by ifenprodil, an inhibitor for polyamine site of NMDAR. The enhancement of LTP by sunifilam was associated with an increase in phosphorylation of α‐amino‐3‐hydroxy‐5‐methylisozazole‐4‐propionate receptor (AMPAR) through activation of calcium/calmodulin‐dependent protein kinase II (CaMKII) and an increase in phosphorylation of NMDAR through activation of protein kinase Cα (PKCα). Sunifiram treatments at 1–1000 nM increased the slope of field excitatory postsynaptic potentials (fEPSPs) in a dose‐dependent manner. The enhancement was associated with an increase in phosphorylation of AMPAR receptor through activation of CaMKII. Interestingly, under the basal condition, sunifiram treatments increased PKCα (Ser‐657) and Src family (Tyr‐416) activities with the same bell‐shaped dose‐response curve as that of LTP peaking at 10 nM. The increase in phosphorylation of PKCα (Ser‐657) and Src (Tyr‐416) induced by sunifiram was inhibited by 7‐ClKN treatment. The LTP enhancement by sunifiram was significantly inhibited by PP2, a Src family inhibitor. Finally, when pretreated with a high concentration of glycine (300 μM), sunifiram treatments failed to potentiate LTP in the CA1 region. Taken together, sunifiram stimulates the glycine‐binding site of NMDAR with concomitant PKCα activation through Src kinase. Enhancement of PKCα activity triggers to potentiate hippocampal LTP through CaMKII activation. © 2013 Wiley Periodicals, Inc.     

Ca2+/calmodulin-dependent protein kinase II and protein kinase C activities mediate extracellular glucose-regulated hippocampal synaptic efficacy

To define how extracellular glucose levels affect synaptic efficacy and long-term potentiation (LTP), we evaluated electrophysiological and neurochemical properties in hippocampal CA1 regions following alterations in glucose levels in the ACSF. In rat hippocampal slices prepared in ACSF with 3.5 mM glucose, fEPSPs generated by Schaffer collateral/commissural stimulation markedly increased when ACSF glucose levels were increased from 3.5 to 7.0 mM. The paired-pulse facilitation reflecting presynaptic transmitter release efficacy was significantly suppressed by elevation to 7.0 mM glucose because of potentiation of the input–output relationship (I/O relationship) of fEPSPs by single pulse stimulation. Prolonged potentiation of fEPSPs by elevation to 7.0 mM glucose coincided with increased autophosphorylation both of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and protein kinase Cα (PKCα). The increased I/O relationship of fEPSPs was also associated with markedly increased synapsin I phosphorylation by CaMKII. Transmitter-evoked postsynaptic currents were also measured in CA1 neurons by electrophoretical application of NMDA and AMPA to the apical dendrites of pyramidal neurons. NMDA- and AMPA-evoked currents were significantly augmented by elevation to 7.0 mM. Notably, high frequency stimulation of the Schaffer collateral/commissural pathway failed to induce LTP in the CA1 region at 3.5 mM glucose but LTP was restored dose-dependently by increasing glucose levels to 7.0 and 10.0 mM. LTP induction in the presence of 7.0 mM glucose was closely associated with further increases in CaMKII autophosphorylation without changes in PKCα autophosphorylation. Taken together, CaMKII and PKC activation likely mediate potentiation of fEPSPs by elevated glucose levels, and CaMKII activity is also associated with LTP induction in the hippocampal CA1 region.     

NMDA receptor blockade prevents the increase in protein kinase C substrate (protein F1) phosphorylation produced by long-term potentiation

Recent evidence has implicated activation of the N-methyl-D-aspartate (NMDA) class of glutamate receptor in the initiation of hippocampal long-term potentiation (LTP), an electrophysiological model of information storage in the brain. A separate line of evidence has suggested that activation of protein kinase C (PKC) and the consequent phosphorylation of its substrates is necessary for the maintenance of the LTP response. To determine if PKC activation is a consequence of NMDA receptor activation during LTP, we applied the NMDA receptor antagonist drug, DL-aminophosphonovalerate (APV) both immediately prior to and following high frequency stimulation, resulting in successful and unsuccessful blockade of LTP initiation, respectively. We then measured the phosphorylation of a PKC substrate (protein F1) in hippocampal tissue dissected from these animals. Only successful blockade of LTP initiation by prior application of APV was seen to block the LTP-associated increase in protein F1 phosphorylation measured in vitro (P less than 0.001 by ANOVA). This suggests that NMDA receptor-mediated initiation triggers maintenance processes that are, at least in part, mediated by protein F1 phosphorylation. These data provide the first evidence linking two mechanisms associated with LTP, NMDA receptor activation and PKC substrate phosphorylation.     

A role for protein kinase C in a form of metaplasticity that regulates the induction of long-term potentiation at CA1 synapses of the adult rat hippocampus

The possibility that protein kinase C (PKC) is involved in the induction of N‐methyl‐ D ‐aspartate (NMDA) receptor‐dependent long‐term potentiation (LTP) at CA1 synapses in the hippocampus has been the subject of considerable investigation. However, many of the conclusions have been drawn from the use of relatively nonspecific PKC inhibitors. In the present study we have examined the role of PKC in tetanus‐induced LTP of AMPA receptor‐mediated synaptic transmission in hippocampal slices obtained from adult rats. In particular, we have investigated the possible role of PKC in a molecular switch process that is triggered by the synaptic activation of metabotropic glutamate receptors and regulates the induction of LTP. We find that the three PKC inhibitors examined, chelerythrine, Ro‐31–8220 and Gö 6983, all block the setting of the molecular switch at concentrations consistent with inhibition of PKC. In contrast, these inhibitors are without affect on the induction of LTP, even when applied in very much higher concentrations. A PKA inhibitor, Rp‐cAMPS, had no effect on either process. We suggest that neither PKC nor PKA is required to induce LTP at this synapse. However, PKC is involved in the regulation of LTP induction, via the molecular switch process.     

Induction and expression of GluA1 (GluR-A)-independent LTP in the hippocampus

Long-term potentiation (LTP) at hippocampal CA3–CA1 synapses is thought to be mediated, at least in part, by an increase in the postsynaptic surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptors induced by N -methyl- d -aspartate (NMDA) receptor activation. While this process was originally attributed to the regulated synaptic insertion of GluA1 (GluR-A) subunit-containing AMPA receptors, recent evidence suggests that regulated synaptic trafficking of GluA2 subunits might also contribute to one or several phases of potentiation. However, it has so far been difficult to separate these two mechanisms experimentally. Here we used genetically modified mice lacking the GluA1 subunit ( Gria1 ^−/− mice) to investigate GluA1-independent mechanisms of LTP at CA3–CA1 synapses in transverse hippocampal slices. An extracellular, paired theta-burst stimulation paradigm induced a robust GluA1-independent form of LTP lacking the early, rapidly decaying component characteristic of LTP in wild-type mice. This GluA1-independent form of LTP was attenuated by inhibitors of neuronal nitric oxide synthase and protein kinase C (PKC), two enzymes known to regulate GluA2 surface expression. Furthermore, the induction of GluA1-independent potentiation required the activation of GluN2B (NR2B) subunit-containing NMDA receptors. Our findings support and extend the evidence that LTP at hippocampal CA3–CA1 synapses comprises a rapidly decaying, GluA1-dependent component and a more sustained, GluA1-independent component, induced and expressed via a separate mechanism involving GluN2B-containing NMDA receptors, neuronal nitric oxide synthase and PKC.     

NMDA receptor–dependent signaling pathways that underlie amyloid β‐protein disruption of LTP in the hippocampus

Alzheimer's disease (AD), the most common neurodegenerative disease in the elderly population, is characterized by the hippocampal deposition of fibrils formed by amyloid β‐protein (Aβ), a 40‐ to 42‐amino‐acid peptide. The folding of Aβ into neurotoxic oligomeric, protofibrillar, and fibrillar assemblies is believed to mediate the key pathologic event in AD. The hippocampus is especially susceptible in AD and early degenerative symptoms include significant deficits in the performance of hippocampal‐dependent cognitive abilities such as spatial learning and memory. Transgenic mouse models of AD that express C‐terminal segments or mutant variants of amyloid precursor protein, the protein from which Aβ is derived, exhibit age‐dependent spatial memory impairment and attenuated long‐term potentiation (LTP) in the hippocampal CA1 and dentate gyrus (DG) regions. Recent experimental evidence suggests that Aβ disturbs N‐methyl‐D ‐aspartic acid (NMDA) receptor–dependent LTP induction in the CA1 and DG both in vivo and in vitro. Furthermore, these studies suggest that Aβ specifically interferes with several major signaling pathways downstream of the NMDA receptor, including the Ca²⁺‐dependent protein phosphatase calcineurin, Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII), protein phosphatase 1, and cAMP response element–binding protein (CREB). The influence of Aβ on each of these downstream effectors of the NMDA receptor is reviewed in this article. Additionally, other mechanisms of LTP modulation, such as Aβ attenuation of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor currents, are briefly discussed. © 2009 Wiley‐Liss, Inc.     

Activation of dopamine D1/D5 receptors facilitates the induction of presynaptic long‐term potentiation at hippocampal output synapses

Encoding of novel information has been proposed to rely on the time‐locked release of dopamine in the hippocampal formation during novelty detection. However, the site of novelty detection in the hippocampus remains a matter of debate. According to current models, the CA1 and the subiculum act as detectors and distributors of novel sensory information. Although most CA1 pyramidal neurons exhibit regular‐spiking behavior, the majority of subicular pyramidal neurons fire high‐frequency bursts of action potentials. The present study investigates the efficacy of dopamine D1/D5 receptor activation to facilitate the induction of activity‐dependent long‐term potentiation (LTP) in rat CA1 regular‐spiking and subicular burst‐spiking pyramidal cells. Using a weak stimulation protocol, set at a level subthreshold for the induction of LTP, we show that activation of D1/D5 receptors for 5–10 min facilitates LTP in subicular burst‐spiking neurons but not in CA1 neurons. The results demonstrate that D1/D5 receptor‐facilitated LTP is NMDA receptor‐dependent, and requires the activation of protein kinase A. In addition, the D1/D5 receptor‐facilitated LTP is shown to be presynaptically expressed and relies on presynaptic Ca²⁺ signaling. The phenomenon of dopamine‐induced facilitation of presynaptic NMDA receptor‐dependent LTP in subicular burst‐spiking pyramidal cells is in accordance with observations of the time‐locked release of dopamine during novelty detection in this brain region, and reveals an intriguing mechanism for the encoding of hippocampal output information.     

Inhibition of protein kinase activity enhances long-term potentiation of hippocampal IPSPs.

In this study on guinea pig hippocampal slices, protein kinase C (PKC) involvement in long-term potentiation (LTP) of GABAA and GABAB receptor-mediated fast and slow IPSPs, respectively, was examined. Stimulation of the stratum radiatum induced EPSPs followed by fast and slow IPSPs in the CA1 neurons. Tetanic stimulation of the stratum caused a marginal LTP of the fast IPSP but not of the slow IPSP. When K-252b, a potent inhibitor of PKC, was injected into CA1 neurons, LTP of fast and slow IPSPs was observed. These results indicate that PKC activation in CA1 neurons is involved in minimizing, rather than inducing, LTP of the IPSPs so that the EPSP is not distorted.     

N-methyl-D-aspartate receptor antagonists are less effective in blocking long-term potentiation at apical than basal dendrites in hippocampal CA1 of awake rats

Long-term potentiation (LTP) of field excitatory postsynaptic potentials (fEPSPs) at the apical or basal dendrites of CA1 pyramidal cells was induced by stimulation with a 1-s train of 200-Hz pulses in awake rats, with or without the presence of various doses of an N-methyl-D-aspartate (NMDA) receptor antagonist. Apical LTP was blocked by an intracerebroventricular (i.c.v.) dose of 40 microg D-2-amino-5-phosphonopentanoic acid (D-AP5) or 20 mg/kg i.p. D-2-amino-4-methyl-5-phosphono-3-pentanoic acid (CGP-40116), whereas basal LTP was blocked by half the dose of D-AP5 or CGP-40116. The noncompetitive antagonist MK-801 (< or =1 mg/kg i.p.) had no significant effect on apical LTP. Apical LTP was not blocked by i.c.v. nifedipine. The effect of an NMDA receptor antagonist alone on apical and basal fEPSPs was also evaluated, to assess the net effect of the NMDA receptor antagonist in blocking LTP. MK-801 (0.5-1 mg/kg i.p.) or CGP-40116 (10-20 mg/kg i.p.) but not D-AP5 suppressed apical fEPSPs for several hours and confounded the expression of apical LTP during this time. We concluded that hippocampal LTP at different synapses has different sensitivity to NMDA receptor antagonists and that a general blockade of hippocampal NMDA receptor functions cannot be inferred by a single hippocampal LTP measure.     

Neurogranin phosphorylation fine-tunes long-term potentiation

Learning-related potentiation of synaptic strength at Cornu ammonis subfield 1 (CA1) hippocampal excitatory synapses is dependent on neuronal activity and the activation of glutamate receptors. However, molecular mechanisms that regulate and fine-tune the expression of long-term potentiation (LTP) are not well understood. Recently it has been indicated that neurogranin (Ng), a neuron-specific, postsynaptic protein that is phosphorylated by protein kinase C, potentiates synaptic transmission in an LTP-like manner. Here, we report that a Ng mutant that is unable to be phosphorylated cannot potentiate synaptic transmission in rat CA1 hippocampal neurons and results in a submaximal expression of LTP. Our results provide the first evidence that the phosphorylation of Ng can regulate LTP expression.     

Critical Role of Presynaptic Nicotinic ACh Receptor in the Formation of Long-Term Potentiation: Implications for Development of Anti-Dementia Drugs

Background : Since neuronal nicotinic ACh receptors are involved in the cognitive function, they have been studied as a target of anti‐dementia drugs. The present study was designed to understand the role of nicotinic ACh receptors in the expression of long‐term potentiation (LTP), a cellular model of learning and memory. Methods : The ultrastructural localization of neuronal nicotinic ACh receptors in the rat hippocampus was examined electron‐immunohistochemically using an antibody against the α7 subunit, forming a brain‐type nicotinic ACh receptor. Miniature excitatory postsynaptic currents (mEPSCs) were monitored in cultured rat hippocampal neurons. Schaffer collateral‐CA1 LTP and perforant path LTP were analyzed by recording field excitatory postsynaptic potentials (fEPSPs) and population spikes (PSs) in the CA1 region and the dentate gyrus of rat hippocampal slices or in the intact mouse hippocampus. Results : α7 receptors are preferentially localized on presynaptic terminals, where the receptors are employed in the release of the excitatory neurotransmitter, glutamate. The probability of LTP development was markedly reduced in the presence of the neuronal nicotinic ACh receptor antagonists, α‐bungarotoxin and mecamylamine, in both the CA1 region and the dentate gyrus of rat hippocampal slices. Perforant path LTP was never induced in slices with selective cholinergic denervation using 192 IgG‐saporin, while it was not affected by atropine, a selective muscarinic ACh receptor antagonist, in normal slices. Nicotine facilitated hippocampal neurotransmission with the saturation occluding the potentiation induced by tetanic stimulation, and vice versa. A similar occlusion was also obtained with an intact mouse hippocampus. These types of LTP, which are dependent upon N‐methyl‐D‐aspartate (NMDA) receptors, were still induced by treatment with nicotine in the presence of D‐2‐amino‐5‐phosphonovaleric acid (APV), a selective NMDA receptor antagonist. Conclusion : The results of the present study suggest that presynaptic nicotinic ACh receptors play a critical role as a target of retrograde messengers in the formation of NMDA receptor‐dependent LTP. This may account for the involvement of nicotinic ACh receptors in cognitive function. Drugs enhancing the activity of neuronal nicotinic ACh receptors, therefore, are capable of expressing LTP, conversely, ameliorating dementia.     

Enhanced phosphorylation of the postsynaptic protein kinase C substrate RC3/neurogranin during long-term potentiation

Long-term potentiation (LTP) is a sustained strengthening of synaptic connections that occurs in the mammalian hippocampus, and is a cellular mechanism likely to contribute to memory formation. One question of current interest is whether the biochemical mechanisms responsible for the maintenance of LTP have a presynaptic or postsynaptic locus. We have determined that the phosphorylation of the postsynaptic protein kinase (PKC) substrate RC3/neurogranin is increased in the maintenance phase of LTP, and that the induction of this effect is dependent on activation of the N-methyl-d-aspartate (NMDA) subtype of glutamate receptors. The sustained increase in RC3/neurogranin phosphorylation requires ongoing protein kinase activity, as application of the protein kinase inhibitor H-7 after LTP induction can reverse the increased RC3/neurogranin phosphorylation. Overall, these data are evidence for postsynaptic biochemical changes in the maintenance of LTP. They also implicate RC3/neurogranin as a downstream effector of PKC activity in LTP that could contribute to physiologic expression of LTP.     

Adenylyl cyclase activation modulates activity-dependent changes in synaptic strength and Ca2+/calmodulin-dependent kinase II autophosphorylation.

Activation of the Ca2+- and calmodulin-dependent protein kinase II (CaMKII) and its conversion into a persistently activated form by autophosphorylation are thought to be crucial events underlying the induction of long-term potentiation (LTP) by increases in postsynaptic Ca2+. Because increases in Ca2+ can also activate protein phosphatases that oppose persistent CaMKII activation, LTP induction may also require activation of signaling pathways that suppress protein phosphatase activation. Because the adenylyl cyclase (AC)-protein kinase A signaling pathway may provide a mechanism for suppressing protein phosphatase activation, we investigated the effects of AC activators on activity-dependent changes in synaptic strength and on levels of autophosphorylated alphaCaMKII (Thr286). In the CA1 region of hippocampal slices, briefly elevating extracellular Ca2+ induced an activity-dependent, transient potentiation of synaptic transmission that could be converted into a persistent potentiation by the addition of phosphatase inhibitors or AC activators. To examine activity-dependent changes in alphaCaMKII autophosphorylation, we replaced electrical presynaptic fiber stimulation with an increase in extracellular K+ to achieve a more global synaptic activation during perfusion of high Ca2+ solutions. In the presence of the AC activator forskolin or the protein phosphatase inhibitor calyculin A, this treatment induced a LTP-like synaptic potentiation and a persistent increase in autophosphorylated alphaCaMKII levels. In the absence of forskolin or calyculin A, it had no lasting effect on synaptic strength and induced a persistent decrease in autophosphorylated alphaCaMKII levels. Our results suggest that AC activation facilitates LTP induction by suppressing protein phosphatases and enabling a persistent increase in the levels of autophosphorylated CaMKII.     

The translocation and involvement of protein kinase C in mossy fiber-CA3 long-term potentiation in hippocampus of the rat brain

The detailed mechanisms underlying long-term potentiation (LTP) are not known. In hippocampal CA1, translocation of protein kinase C (PKC) activity from cytosol to membrane and subsequent phosphorylation of growth associated protein (GAP)-43 have been demonstrated to be critical events for the maintenance phase of LTP. LTP in mossy fiber (MF)-CA3 pathway and the Schaffer collateral/commissural (SC)-CA1 pathway differ in a number of ways: SC-CA1 LTP depends on NMDA receptors while MF-CA3 LTP does not, and SC-CA1 LTP is primarily postsynaptic while MF-CA3 LTP is primarily presynaptic. The role of PKC in MF-CA3 LTP has not been studied. We investigated the role of PKC in CA3 and show that PKC inhibitors prevent LTP, but that PKC activators produce a reversible synaptic potentiation, indicating that PKC activation is an essential but not sufficient component of LTP in CA3. Then using antibodies against specific PKC isozymes we have determined the membrane vs. cytosolic distribution of various PKC isozymes in slices subjected to low or tetanic stimulation, or perfused with phorbol esters (PDAc). Compared with control, LTP and PDAc slices show greater PKC-α and -ε immunoreactivity in the membrane fraction, indicating that both LTP and phorbol ester treatment induce translocation of PKC-α and -ε from cytosol to membrane. However, with PKC-β and PKC-γ the only detectable translocation from cytosol to membrane was in the phorbol ester-treated slices. Thus, while phorbol ester treatment causes translocation of PKC-α, -β, -γ and -ε, the only detectable translocation associated with CA3 LTP is that of PKC-α and -ε.     

Different mechanisms may be required for maintenance,of NMDA receptor-dependent and independent forms of long-term potentiation

In hippocampal area CA1, long-term potentiation (LTP) is induced by tetanic stimulation protocols that activate N-methyl-D-aspartate (NMDA) receptors. In addition, some stimulation protocols can induce LTP during NMDA receptor blockade. An initial signal in both NMDA receptor-dependent and independent LTPs is increased intracellular Ca²⁺ concentration in postsynaptic neurons. It therefore seems possible that subsequent steps leading to expression and maintenance of potentiation are shared whether or not LTP is induced through NMDA receptor activation. We tested this hypothesis by applying a broad spectrum protein kinase, inhibitor, previously shown to inhibit NMDA receptor-dependent LTP. In agreement with earlier reports, we found that H-7 inhibited NMDA receptor-dependent LTP when applied either during tetanic stimulation, or beginning 30 min following tetanic stimulation. In contrast, NMDA receptorindependent LTP was not inhibited by H-7 applied during or following tetanic stimulation. We also tested for mutual occlusion between NMDA receptor-dependent and independent LTPs. Although induction of NMDA receptor-independent LTP did not occlude later induction of NMDA receptor-dependent LTP, induction of NMDA receptordependent LTP did occlude NMDA receptor-independent LTP. While the kinase inhibitor experiment showed a clear difference between NMDA receptor-dependent and independent LTPs, the occlusion experiments suggest an interaction between the signalling pathways for the two LTPs. © 1995 Wiley-Liss, Inc.     

The MUPP1-SynGAPalpha protein complex does not mediate activity-induced LTP

At excitatory synapses of hippocampal neurons, the multi-PDZ domain scaffolding protein, MUPP1, assembles the NR2B subunit of the NMDA receptor (NMDAR), Ca2+-calmodulin kinase (CamKII), and the alpha1 isoform of the postsynaptic density GTPase activating protein, SynGAP (SynGAPalpha). In order to evaluate the role of this complex in excitatory synaptic neurotransmission we specifically disrupted MUPP1-SynGAPalpha interactions in CA1 neurons of acute hippocampal slices using intracellular perfusion with peptides derived from SynGAPalpha-MUPP1 binding domains. Disruption of the interaction between MUPP1 and SynGAPalpha with two complementary peptides derived from SynGAP and MUPP1 mutual binding sites resulted in enhancement of excitatory postsynaptic currents (EPSCs). This potentiation did not occlude pairing-induced long-term potentiation (LTP); indeed the amplitude of postsynaptic responses of activity-potentiated synapses was further increased. Pre-potentiation of excitatory synapses with theta burst stimulations did not modify the MUPP1-SynGAPalpha-dependent enhancement of EPSCs. Our data suggest that MUPP1-SynGAPalpha complex dissociation triggers a mechanism for AMPAR enhancement that is distinct from activity-induced LTP.     

Prepubertal castration causes the age‐dependent changes in hippocampal long‐term potentiation

The effects of prepubertal castration on hippocampal CA3‐CA1 synaptic transmission and plasticity were studied at different ages in vitro. The field excitatory postsynaptic potentials (fEPSP) and population spikes (PS) were simultaneously recorded from stratum radiatum and stratum pylamidale of area CA1 following stimulation of Schaffer collaterals in slices taken from sham‐castrated and castrated rats at postnatal days (PND) 28, 35, 45, and 60. Castration had no effect on baseline responses at different ages except at PND 60 that a decrease in the fEPSP slope was seen. Prepubertal castration caused age‐specific changes in CA1‐long term potentiation (LTP) induction. The castration did decrease both fEPSP‐LTP and PS‐LTP at PND 35 but a decrease was seen only in PS‐LTP at PND 60. NMDA receptor antagonist AP5 (25 µM) completely blocked both fEPSP‐LTP and PS‐LTP at PND 60 and only PS‐LTP at PND 35 in both sham‐castrated and castrated groups. Although AP5 blocked fEPSP‐LTP at PND 35 in sham‐castrated group, it failed to inhibit fEPSP‐LTP at PND 35 in castrated one. These findings suggest that prepubertal castration causes the age‐dependent changes in CA1‐LTP induction, which might arise from alterations in the NMDA receptors. Synapse 67:235–244, 2013. © 2013 Wiley Periodicals, Inc.     

Involvement of PKA and ERK pathways in ghrelin‐induced long‐lasting potentiation of excitatory synaptic transmission in the CA1 area of rat hippocampus

Acute effects of ghrelin on excitatory synaptic transmission were evaluated on hippocampal CA1 synapses. Ghrelin triggered an enduring enhancement of synaptic transmission independently of NMDA receptor activation and probably via postsynaptic modifications. This ghrelin‐mediated potentiation resulted from the activation of GHS‐R1a receptors as it was mimicked by the selective agonist JMV1843 and blocked by the selective antagonist JMV2959. This potentiation also required the activation of PKA and ERK pathways to occur as it was inhibited by KT5720 and U0126, respectively. Moreover it most probably involved Ca²⁺ influxes as both ghrelin and JMV1843 elicited intracellular Ca²⁺ increases, which were dependent on the presence of extracellular Ca²⁺ and mediated by L‐type Ca²⁺ channels opening. In addition, ghrelin potentiated AMPA receptor‐mediated [Ca²⁺]i increases while decreasing NMDA receptor‐mediated ones. Thus the potentiation of synaptic transmission by GHS‐R1a at hippocampal CA1 excitatory synapses probably results from postsynaptic mechanisms involving PKA and ERK activation, which are producing long‐lasting enhancement of AMPA receptor‐mediated responses.     

Estrogen induces a rapid increase of calcium-calmodulin-dependent protein kinase II activity in the hippocampus

Molecular genetics experiments using gene targeting and transgenic technology demonstrated the importance of -calcium-calmodulin-dependent protein kinase II (CaMKII) in long-term potentiation (LTP) and memory. Little information is available though on how CaMKII activity may be regulated in vivo. We show that estradiol benzoate activates CaMKII in a dose and time-dependent manner in mouse hippocampus after 30 min stimulation. The effect of estrogen is via a very rapid nongenomic mechanism that is blocked in vitro in hippocampal primary neurons by the pure estrogen receptor antagonist, ICI 182,780. These results suggest that estrogen action in the hippocampus is linked to CaMKII activation.     

Enhanced activation of Ca2+/calmodulin-dependent protein kinase II upon downregulation of cyclin-dependent kinase 5-p35

Cyclin-dependent kinase 5 (Cdk5)-p35 is downregulated in cultured neurons by N-methyl-D-aspartate (NMDA) via the proteasomal degradation of p35. However, it is not known where in neurons this downregulation occurs or the physiologic meaning of the reaction. We show the enrichment of Cdk5 and p35 in the postsynaptic density and the NMDA-induced degradation of postsynaptic p35 using brain slices and cultured neurons. To evaluate the role of this downregulation, we examined the relationship between Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activation and Cdk5 downregulation, as events downstream from NMDA stimulation. Glutamate or NMDA stimulation induced CaMKII autophosphorylation over a time course that mirrored the time course of p35 degradation. To simulate the downregulation of postsynaptic Cdk5 in invitro experiments, we used the Cdk5 inhibitor roscovitine. The inhibition of Cdk5 activity by roscovitine enhanced CaMKII autophosphorylation and activation in cultured neurons, and in an isolated postsynaptic-density-enriched fraction. These results suggest that Cdk5 activity suppresses CaMKII activation, and that the downregulation of Cdk5 activity after treatment withNMDA facilitates CaMKII activation, leading to the easier induction of long-term potentiation.