Search

Endocannabinoids (eCBs), including AEA and 2-AG, are endogenous signaling mediators involved in many physiological and pathological events. The G protein-coupled cannabinoid receptor 1 (CB1 R) is an important target for eCBs, however, additional non-CB1 receptor targets have also been identified. Although recent evidence suggests that NMDA receptor function may be regulated by eCBs, the underlying mechanisms remain poorly characterized. Using acutely isolated CA1 neurons and slices from the hippocampus, we found that both AEA and 2-AG potentiate NMDAR-mediated currents independently of CB1 receptors (CB1 Rs) and via distinct signaling pathways. Potentiation by AEA requires the activation of TRPV1 channels. In contrast, potentiation by 2-AG requires the sequential activation of PKC and Src. Additionally, in hippocampal slices, we found that both AEA and 2-AG induce NMDAR-mediated metaplasticity and facilitate the induction of subsequent LTD independently of CB1 Rs. Enhanced LTD by AEA, but not 2-AG, was dependent on TRPV1 channels. Our findings reveal previously unrecognized non-CB1 R-dependent signaling cascades through which the two major eCBs regulate NMDA receptor function and consequently synaptic plasticity., (© 2014 Wiley Periodicals, Inc.)

Background: Excess expression of acetylcholinesterase (AChE) in the cortex and hippocampus causes a decrease in the number of glutamatergic synapses and alters the expression of neurexin and neuroligin, trans-synaptic proteins that control synaptic stability. The molecular sequence and three-dimensional structure of AChE are homologous to the corresponding aspects of the ectodomain of neuroligin. This study investigated whether excess AChE interacts physically with neurexin to destabilize glutamatergic synapses., Results: The results showed that AChE clusters colocalized with neurexin assemblies in the neurites of hippocampal neurons and that AChE co-immunoprecipitated with neurexin from the lysate of these neurons. Moreover, when expressed in human embryonic kidney 293 cells, N-glycosylated AChE co-immunoprecipitated with non-O-glycosylated neurexin-1β, with N-glycosylation of the AChE being required for this co-precipitation to occur. Increasing extracellular AChE decreased the association of neurexin with neuroligin and inhibited neuroligin-induced synaptogenesis. The number and activity of excitatory synapses in cultured hippocampal neurons were reduced by extracellular catalytically inactive AChE., Conclusions: Excessive glycosylated AChE could competitively disrupt a subset of the neurexin-neuroligin junctions consequently impairing the integrity of glutamatergic synapses. This might serve a molecular mechanism of excessive AChE induced neurodegeneration.

In Alzheimer's disease (AD), soluble amyloid-β oligomers (AβOs) trigger neurotoxic signaling, at least partially, via the cellular prion protein (PrP(C)). However, it is unknown whether other ligands of PrP(C) can regulate this potentially toxic interaction. Stress-inducible phosphoprotein 1 (STI1), an Hsp90 cochaperone secreted by astrocytes, binds to PrP(C) in the vicinity of the AβO binding site to protect neurons against toxic stimuli. Here, we investigated a potential role of STI1 in AβO toxicity. We confirmed the specific binding of AβOs and STI1 to the PrP and showed that STI1 efficiently inhibited AβO binding to PrP in vitro (IC50 of ∼70 nm) and also decreased AβO binding to cultured mouse primary hippocampal neurons. Treatment with STI1 prevented AβO-induced synaptic loss and neuronal death in mouse cultured neurons and long-term potentiation inhibition in mouse hippocampal slices. Interestingly, STI1-haploinsufficient neurons were more sensitive to AβO-induced cell death and could be rescued by treatment with recombinant STI1. Noteworthy, both AβO binding to PrP(C) and PrP(C)-dependent AβO toxicity were inhibited by TPR2A, the PrP(C)-interacting domain of STI1. Additionally, PrP(C)-STI1 engagement activated α7 nicotinic acetylcholine receptors, which participated in neuroprotection against AβO-induced toxicity. We found an age-dependent upregulation of cortical STI1 in the APPswe/PS1dE9 mouse model of AD and in the brains of AD-affected individuals, suggesting a compensatory response. Our findings reveal a previously unrecognized role of the PrP(C) ligand STI1 in protecting neurons in AD and suggest a novel pathway that may help to offset AβO-induced toxicity.

Background: N-methyl-D-aspartate (NMDA) receptors are regulated by several G protein-coupled receptors (GPCRs) as well as receptor tyrosine kinases. Serotonin (5-HT) type 7 receptors are expressed throughout the brain including the thalamus and hippocampus. Long-term (2-24 h) activation of 5-HT7 receptors promotes the expression of neuroprotective growth factor receptors, including the platelet-derived growth factor (PDGF) β receptors which can protect neurons against NMDA-induced neurotoxicity., Results: In contrast to long-term activation of 5-HT7 receptors, acute (5 min) treatment of isolated hippocampal neurons with the 5-HT7 receptor agonist 5-carboxamidotryptamine (5-CT) enhances NMDA-evoked peak currents and this increase in peak currents is blocked by the 5-HT7 receptor antagonist, SB 269970. In hippocampal slices, acute 5-HT7 receptor activation increases NR1 NMDA receptor subunit phosphorylation and differentially alters the phosphorylation state of the NR2B and NR2A subunits. NMDA receptor subunit cell surface expression is also differentially altered by 5-HT7 receptor agonists: NR2B cell surface expression is decreased whereas NR1 and NR2A surface expression are not significantly altered., Conclusions: In contrast to the negative regulatory effects of long-term activation of 5-HT7 receptors on NMDA receptor signaling, acute activation of 5-HT7 receptors promotes NMDA receptor activity. These findings highlight the potential for temporally differential regulation of NMDA receptors by the 5-HT7 receptor.

Background: Glutathione (GSH) plays an important role in neuronal oxidant defence. Depletion of cellular GSH is observed in neurodegenerative diseases and thereby contributes to the associated oxidative stress and Ca2+ dysregulation. Whether depletion of cellular GSH, associated with neuronal senescence, directly influences Ca2+ permeation pathways is not known. Transient receptor potential melastatin type 2 (TRPM2) is a Ca2+ permeable non-selective cation channel expressed in several cell types including hippocampal pyramidal neurons. Moreover, activation of TRPM2 during oxidative stress has been linked to cell death. Importantly, GSH has been reported to inhibit TRPM2 channels, suggesting they may directly contribute to Ca2+ dysregulation associated with neuronal senescence. Herein, we explore the relation between cellular GSH and TRPM2 channel activity in long-term cultures of hippocampal neurons., Results: In whole-cell voltage-clamp recordings, we observe that TRPM2 current density increases in cultured pyramidal neurons over time in vitro. The observed increase in current density was prevented by treatment with NAC, a precursor to GSH synthesis. Conversely, treatment of cultures maintained for 2 weeks in vitro with L-BSO, which depletes GSH by inhibiting its synthesis, augments TRPM2 currents. Additionally, we demonstrate that GSH inhibits TRPM2 currents through a thiol-independent mechanism, and produces a 3.5-fold shift in the dose-response curve generated by ADPR, the intracellular agonist for TRPM2., Conclusion: These results indicate that GSH plays a physiologically relevant role in the regulation of TRPM2 currents in hippocampal pyramidal neurons. This interaction may play an important role in aging and neurological diseases associated with depletion of GSH.

Transplantation of neural stem/progenitor cells (NSPCs) is a promising strategy for repair of the diseased/injured central nervous system (CNS); however, controlling their differentiation remains a significant hurdle. This study is aimed at controlling differentiation and specifically at screening exogenous factors to direct NSPC differentiation into neurons in vitro. In this study, adult rat SVZ-derived NSPCs were treated with several factors and screened individually and in combination for changes in cellular morphology, neuronal marker expression, quantitative real-time qRT-PCR, and electrophysiological properties. These in vitro screens showed that of all the different treatments, dibutyryl cyclic AMP (dbcAMP) and interferon-gamma (IFN-gamma) enhanced neuronal differentiation most significantly compared to the 1% fetal bovine serum (FBS) controls. Importantly, the combined treatment of NSPCs with dbcAMP and IFN-gamma promoted greater neuronal differentiation as reflected by an increase in beta-III tubulin expression and morphological differentiation. Interestingly, the neurons that were generated from the NSPCs in vitro in the presence of dbcAMP and IFN-gamma, alone or in combination, responded to exogenous glutamate (Glu), but not gamma-aminobutyric acid (GABA), indicating that these neurons express glutamate receptors. These NSPC-derived neurons may be promising for neural regenerative strategies in the CNS.

Cardiac arrest victims may experience transient brain hypoperfusion leading to delayed death of hippocampal CA1 neurons and cognitive impairment. We prevented this in adult rats by inhibiting the expression of transient receptor potential melastatin 7 (TRPM7), a transient receptor potential channel that is essential for embryonic development, is necessary for cell survival and trace ion homeostasis in vitro, and whose global deletion in mice is lethal. TRPM7 was suppressed in CA1 neurons by intrahippocampal injections of viral vectors bearing shRNA specific for TRPM7. This had no ill effect on animal survival, neuronal and dendritic morphology, neuronal excitability, or synaptic plasticity, as exemplified by robust long-term potentiation (LTP). However, TRPM7 suppression made neurons resistant to ischemic death after brain ischemia and preserved neuronal morphology and function. Also, it prevented ischemia-induced deficits in LTP and preserved performance in fear-associated and spatial-navigational memory tasks. Thus, regional suppression of TRPM7 is feasible, well tolerated and inhibits delayed neuronal death in vivo.

Vasoactive intestinal peptide (VIP) is a 28-amino acid peptide, which belongs to a superfamily of structurally related peptide hormones including pituitary adenylate cyclase-activating polypeptide (PACAP). Although several studies have identified the involvement of PACAP in learning and memory, little work has been done to investigate such a role for VIP. At least three receptors for VIP have been identified including the PACAP receptor (PAC1-R) and the two VIP receptors (VPAC receptors). VIP can activate the PAC1-R only if it is used at relatively high concentrations (e.g., 100 nM); however, at lower concentrations (e.g., 1 nM) it is selective for the VPAC receptors. Our lab has showed that PAC1-R activation signals through PKC/CAKbeta/Src pathway to regulate NMDA receptors; however, there is little known about the potential regulation of NMDA receptors by VPAC receptors. Our studies demonstrated that application of 1 nM VIP enhanced NMDA currents by stimulating the VPAC receptors as the effect was blocked by VPAC receptor antagonist [Ac-Tyr(1), D-Phe(2)]GRF (1-29). This enhancement of NMDA currents was blocked by both Rp-cAMPS and PKI(14-22) (they are highly specific PKA inhibitors), but not by the specific PKC inhibitor, bisindolylmaleimide I. In addition, the VIP-induced enhancement of NMDA currents was accentuated by inhibition of phosphodiesterase 4, which inhibits the degradation of cAMP. This regulation of NMDA receptors also required the scaffolding protein AKAP. In contrast, the potentiation induced by high concentration of VIP (e.g., 100 nM) was mediated by PAC1-R as well as by Src kinase. Overall, these results show that VIP can regulate NMDA receptors through different receptors and signaling pathways., ((c) 2009 Wiley-Liss, Inc.)

Platelet-derived growth factor (PDGF) beta receptor activation inhibits N-methyl-d-aspartate (NMDA)-evoked currents in hippocampal and cortical neurons via the activation of phospholipase Cgamma, PKC, the release of intracellular calcium, and a rearrangement of the actin cytoskeleton. In the hippocampus, the majority of NMDA receptors are heteromeric; most are composed of 2 NR1 subunits and 2 NR2A or 2 NR2B subunits. Using NR2B- and NR2A-specific antagonists, we demonstrate that PDGF-BB treatment preferentially inhibits NR2B-containing NMDA receptor currents in CA1 hippocampal neurons and enhances long-term depression in an NR2B subunit-dependent manner. Furthermore, treatment of hippocampal slices or cultures with PDGF-BB decreases the surface localization of NR2B but not of NR2A subunits. PDGFbeta receptors colocalize to a higher degree with NR2B subunits than with NR2A subunits. After neuronal injury, PDGFbeta receptors and PDGF-BB are up-regulated and PDGFbeta receptor activation is neuroprotective against glutamate-induced neuronal damage in cultured neurons. We demonstrate that the neuroprotective effects of PDGF-BB are occluded by the NR2B antagonist, Ro25-6981, and that PDGF-BB promotes NMDA signaling to CREB and ERK1/2. We conclude that PDGFbetaR signaling, by preferentially targeting NR2B receptors, provides an important mechanism for neuroprotection by growth factors in the central nervous system.

TRPM2 is a Ca(2+)-permeable member of the transient receptor potential melastatin family of cation channels whose activation by reactive oxygen/nitrogen species (ROS/RNS) and ADP-ribose (ADPR) is linked to cell death. While these channels are broadly expressed in the CNS, the presence of TRPM2 in neurons remains controversial and more specifically, whether they are expressed in neurons of the hippocampus is an open question. With this in mind, we examined whether functional TRPM2 channels are expressed in this neuronal population. Using a combination of molecular and biochemical approaches, we demonstrated the expression of TRPM2 transcripts and proteins in hippocampal pyramidal neurons. Whole-cell voltage-clamp recordings were subsequently carried out to assess the presence of TRPM2-mediated currents. Application of hydrogen peroxide or peroxynitrite to cultured hippocampal pyramidal neurons activated an inward current that was abolished upon removal of extracellular Ca(2+), a hallmark of TRPM2 activation. When ADPR (300 microM) was included in the patch pipette, a large inward current developed but only when depolarizing voltage ramps were continuously (1/10 s) applied to the membrane. This current exhibited a linear current-voltage relationship and was sensitive to block by TRPM2 antagonists (i.e. clotrimazole, flufenamic acid and N-(p-amylcinnamoyl)anthranilic acid (ACA)). The inductive effect of voltage ramps on the ADPR-dependent current required voltage-dependent Ca(2+) channels (VDCCs) and a rise in [Ca(2+)](i). Consistent with the need for a rise in [Ca(2+)](i), activation of NMDA receptors (NMDARs), which are highly permeable to Ca(2+), was also permissive for current development. Importantly, given the prominent vulnerability of CA1 neurons to free-radical-induced cell death, we confirmed that, with ADPR in the pipette, a brief application of NMDA could evoke a large inward current in CA1 pyramidal neurons from hippocampal slices that was abolished by the removal of extracellular Ca(2+), consistent with TRPM2 activation. Such a current was absent in interneurons of CA1 stratum radiatum. Finally, infection of cultured hippocampal neurons with a TRPM2-specific short hairpin RNA (shRNA(TRPM2)) significantly reduced both the expression of TRPM2 and the amplitude of the ADPR-dependent current. Taken together, these results indicate that hippocampal pyramidal neurons possess functional TRPM2 channels whose activation by ADPR is functionally coupled to VDCCs and NMDARs through a rise in [Ca(2+)](i).

Background: We have previously demonstrated that PDGF receptor activation indirectly inhibits N-methyl-D-aspartate (NMDA) currents by modifying the cytoskeleton. PDGF receptor ligand is also neuroprotective in hippocampal slices and cultured neurons. PDGF receptors are tyrosine kinases that control a variety of signal transduction pathways including those mediated by PLCγ. In fibroblasts Src and another non-receptor tyrosine kinase, Abelson kinase (Abl), control PDGF receptor regulation of cytoskeletal dynamics. The mechanism whereby PDGF receptor regulates cytoskeletal dynamics in central neurons remains poorly understood., Results: Intracellular applications of active Abl, but not heat-inactivated Abl, decreased NMDA-evoked currents in isolated hippocampal neurons. This mimics the effects of PDGF receptor activation in these neurons. The Abl kinase inhibitor, STI571, blocked the inhibition of NMDA currents by Abl. We demonstrate that PDGF receptors can activate Abl kinase in hippocampal neurons via mechanisms similar to those observed previously in fibroblasts. Furthermore, PDGFβ receptor activation alters the subcellular localization of Abl. Abl kinase is linked to actin cytoskeletal dynamics in many systems. We show that the inhibition of NMDA receptor currents by Abl kinase is blocked by the inclusion of the Rho kinase inhibitor, Y-27632, and that activation of Abl correlates with an increase in ROCK tyrosine phosphorylation., Conclusion: This study demonstrates that PDGFβ receptors act via an interaction with Abl kinase and Rho kinase to regulated cytoskeletal regulation of NMDA receptor channels in CA1 pyramidal neurons.

The induction of long-term potentiation at CA3-CA1 synapses is caused by an N-methyl-d-aspartate (NMDA) receptordependent accumulation of intracellular Ca(2+), followed by Src family kinase activation and a positive feedback enhancement of NMDA receptors (NMDARs). Nevertheless, the amplitude of baseline transmission remains remarkably constant even though low frequency stimulation is also associated with an NMDAR-dependent influx of Ca(2+) into dendritic spines. We show here that an interaction between C-terminal Src kinase (Csk) and NMDARs controls the Src-dependent regulation of NMDAR activity. Csk associates with the NMDAR signaling complex in the adult brain, inhibiting the Src-dependent potentiation of NMDARs in CA1 neurons and attenuating the Src-dependent induction of long-term potentiation. Csk associates directly with Src-phosphorylated NR2 subunits in vitro. An inhibitory antibody for Csk disrupts this physical association, potentiates NMDAR mediated excitatory postsynaptic currents, and induces long-term potentiation at CA3-CA1 synapses. Thus, Csk serves to maintain the constancy of baseline excitatory synaptic transmission by inhibiting Src kinase-dependent synaptic plasticity in the hippocampus.

Exposure to low Ca(2+) and/or Mg(2+) is tolerated by cardiac myocytes, astrocytes, and neurons, but restoration to normal divalent cation levels paradoxically causes Ca(2+) overload and cell death. This phenomenon has been called the "Ca(2+) paradox" of ischemia-reperfusion. The mechanism by which a decrease in extracellular Ca(2+) and Mg(2+) is "detected" and triggers subsequent cell death is unknown. Transient periods of brain ischemia are characterized by substantial decreases in extracellular Ca(2+) and Mg(2+) that mimic the initial condition of the Ca(2+) paradox. In CA1 hippocampal neurons, lowering extracellular divalents stimulates a nonselective cation current. We show that this current resembles TRPM7 currents in several ways. Both (i) respond to transient decreases in extracellular divalents with inward currents and cell excitation, (ii) demonstrate outward rectification that depends on the presence of extracellular divalents, (iii) are inhibited by physiological concentrations of intracellular Mg(2+), (iv) are enhanced by intracellular phosphatidylinositol 4,5-bisphosphate (PIP(2)), and (v) can be inhibited by Galphaq-linked G protein-coupled receptors linked to phospholipase C beta1-induced hydrolysis of PIP(2). Furthermore, suppression of TRPM7 expression in hippocampal neurons strongly depressed the inward currents evoked by lowering extracellular divalents. Finally, we show that activation of TRPM7 channels by lowering divalents significantly contributes to cell death. Together, the results demonstrate that TRPM7 contributes to the mechanism by which hippocampal neurons "detect" reductions in extracellular divalents and provide a means by which TRPM7 contributes to neuronal death during transient brain ischemia.

Cultured neurons have been used for investigating synaptic plasticity due to their accessibility to biochemical and immunochemical assays. In addition, recording spontaneous postsynaptic miniature events provides important information about the mechanisms involved in the modulation of synaptic transmission. To automatically detect the spontaneous events, we developed a technique in which a weighted least squares algorithm was used instead of an ordinary one. In addition, multiple templates were used simultaneously to scan the data to increase the accuracy of the fit between the data and templates. An important outcome of the weighted template technique is that the detection rate is not as sensitive to the length of templates and this technique is capable of detection overlapping events reliably.

The excitotoxic theory of stroke, which implicated N-methyl-d-aspartate (NMDA) receptors as mediators of excessive Ca(2+) entry and neuronal death, generated a great deal of enthusiasm for the prospect of using NMDA receptor antagonists to prevent the associated brain injury. Unfortunately, these receptor antagonists failed to provide effective treatments for human stroke. In part, the failure is likely a consequence of having to administer these drugs within a very short therapeutic window after stroke and to the intolerable psychomimetic side effects associated with their use. However, new possibilities for therapeutic intervention are revealing themselves as our understanding of excitotoxicity evolves. We now recognize that ischemia and Ca(2+) toxicity in central neurons can be attributed to a variety of mechanisms recruited downstream of NMDA receptor activation. These include the activation of Ca(2+)-permeable transient receptor potential channels of the melastatin family. The more-delayed activation of these channels offers the tantalizing possibility that drugs targeting selected members of this family may possess a wider therapeutic window for preventing the debilitating consequences after stroke onset.

NMDA receptor function is modulated by both G-protein-coupled receptors and receptor tyrosine kinases. In acutely isolated rat hippocampal neurons, direct activation of the platelet-derived growth factor (PDGF) receptor or transactivation of the PDGF receptor by D4 dopamine receptors inhibits NMDA-evoked currents in a phospholipase C (PLC)-dependent manner. We have investigated further the ability of D2-class dopamine receptors to modulate NMDA-evoked currents in isolated rat prefrontal cortex (PFC). We have demonstrated that, similar to isolated hippocampal neurons, the application of PDGF-BB or quinpirole to isolated PFC neurons induces a slow-onset and long-lasting inhibition of NMDA-evoked currents. However, in contrast to hippocampal neurons, the inhibition of NMDA-evoked currents by quinpirole in PFC neurons is dependent upon D2/3, rather than D4, dopamine receptors. In PFC slices, application of both PDGF-BB and quinpirole induced a phosphorylation of the PDGF receptor at the PLCgamma binding and activation site, Tyr1021. The PDGF receptor kinase inhibitor, tyrphostin A9, and the D2/3 dopamine receptor antagonist, raclopride, inhibited quinpirole-induced Tyr1021 phosphorylation. These finding suggest that quinpirole treatment inhibits NMDAR signaling via PDGF receptor transactivation in both the hippocampus and the PFC, and that the effects of quinpirole in these regions are mediated by D4 and D2/3 dopamine receptors, respectively.

Traumatic brain injury causes neuronal disruption and triggers secondary events leading to additional neuronal death. To study injuries triggered by secondary events, we exposed cultured cortical neurons to sublethal mechanical stretch, thus eliminating confounding death from primary trauma. Sublethally stretched neurons maintained cell membrane integrity, viability, and electrophysiological function. However, stretching induced in the cells a heightened vulnerability to subsequent challenges with L-glutamate or NMDA. This heightened vulnerability was specifically mediated by NMDA receptors (NMDARs), as stretched neurons did not become more vulnerable to either kainate toxicity or to that induced by the Ca(2+) ionophore A23187. Stretch-enhanced vulnerability to NMDA occurred independently of endogenous glutamate release, but required Ca(2+) and Na(+) influx through NMDARs. Stretch did not affect the electrophysiological properties of NMDARs nor excitatory synaptic activity, indicating that specificity of enhanced vulnerability to NMDA involves postsynaptic mechanisms downstream from NMDARs. To test whether this specificity requires physical interactions between NMDARs and cytoskeletal elements, we perturbed actin filaments and microtubules, both of which are linked to NMDARs. This had no effect on the stretch-induced vulnerability to NMDA, suggesting that sublethal stretch does not affect cell survival through the cytoskeleton. Our data illustrate that sublethal in vitro stretch injury triggers distinct signaling pathways that lead to secondary injury, rather than causing a generalized increase in vulnerability to secondary insults.

Although the mechanisms underlying the use-dependent inhibition of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) by barbiturates are not well understood, it has generally been assumed to involve open channel block. We examined the properties of the inhibition of AMPARs by the barbiturate pentobarbital (PB) in acutely isolated and cultured hippocampal neurons. PB caused a use- and concentration-dependent inhibition (IC50 = 20.7 microM) of AMPAR-mediated currents evoked by kainate. Contrary to the properties of an open channel blocker, the inhibition by PB developed with double exponential kinetics was reduced under conditions that favor the open channel state of AMPARs and was independent of membrane voltage. In addition, the inhibition was reduced at basic pH, indicating that the uncharged form of PB is active at AMPARs. Preventing AMPAR desensitization with cyclothiazide reduced the potency of inhibition by PB and prevented its trapping after the removal of agonist. PB preferentially reduced the steady-state (IC50 = 92.8 microM), rather than peak (IC50 > 1 mM) component of responses evoked by glutamate and accelerated the onset of desensitization in a concentration-dependent manner. Miniature excitatory postsynaptic currents recorded from cultured hippocampal neurons, the time course of which is minimally influenced by desensitization, are not inhibited by PB. The sensitivity of AMPAR-mediated synaptic responses to inhibition by PB therefore depends on the contribution of desensitization to these events. Our results suggest that PB does not act as an open channel blocker of AMPARs. Rather, the sensitivity, use dependence, and trapping of inhibition by PB are determined by AMPARs desensitization.

In the central nervous system, excitatory synaptic transmission is mediated by the neurotransmitter glutamate and its receptors. Interestingly, stimulation of group I metabotropic glutamate receptors (mGluRs) can either enhance or depress synaptic transmission at CA1 hippocampal synapses. Here we report that co-activation of mGluR5, a member of the group I mGluR family, and N-methyl-d-aspartate receptors (NMDARs) potentiates NMDAR currents and induces a long lasting enhancement of excitatory synaptic transmission in primary cultured hippocampal neurons. Unexpectedly, activation of mGluR5 alone fails to enhance evoked NMDAR currents and synaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) AMPAR currents. The observed potentiation requires an mGluR5-induced, inositol 1,4,5-trisphosphate receptor-mediated mobilization of intracellular Ca2+, which acts in concert with a protein kinase C, calcium-activated tyrosine kinase cascade to induce a long lasting enhancement of NMDAR and AMPAR currents.

The effects of extracellular pH (pHo) on calcium-sensing non-selective cation (csNSC) channels in cultured mouse hippocampal neurons were investigated using whole-cell voltage-clamp and current-clamp recordings. Decreasing extracellular Ca2+ concentrations ([Ca2+]o) activated slow and sustained inward currents through the csNSC channels. Decreasing pHo activated amiloride-sensitive transient proton-gated currents which decayed to baseline in several seconds. With proton-gated channels inactivated by pre-perfusion with low pH solution or blocked by amiloride, decreasing pHo to 6.5 inhibited the csNSC currents with a leftward shift of the Ca2+ dose-inhibition curve. Increasing pH to 8.5, on the other hand, caused a rightward shift of the Ca2+ dose-inhibition curve and potentiated the csNSC currents. Intracellular alkalinization following bath perfusion of quinine mimicked the potentiation of the csNSC currents by increasing pHo, while intracellular acidification by addition and subsequent withdrawal of NH4Cl mimicked the inhibition of the csNSC currents by decreasing pHo. Intracellular pH (pHi) imaging demonstrated that decreasing pHo induced a corresponding decrease in pHi. Including 30 mM Hepes in the pipette solution eliminated the effects of quinine and NH4Cl on the csNSC currents, but only partially reduced the effect of lowering pHo. In current-clamp recordings, decreasing [Ca2+]o induced sustained membrane depolarization and excitation of hippocampal neurons. Decreasing pHo to 6.5 inhibited the low [Ca2+]o-induced csNSC channel-mediated membrane depolarization and the excitation of neurons. Our results indicate that acidosis may inhibit low [Ca2+]o-induced neuronal excitation by inhibiting the activity of the csNSC channels. Both the extracellular and the intracellular sites are involved in the proton modulation of the csNSC channels.

Hippocampal CA1 homosynaptic long-term potentiation (LTP) is expressed specifically at activated synapses. Increased insertion of postsynaptic alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptors (AMPARs) appears to be crucial for CA1 LTP. However, the mechanism underlying AMPAR insertion during LTP remains largely unknown. We now report that phosphatidylinositol 3-kinase (PI3K) is complexed with AMPARs at synapses and activated by selective stimulation of synaptic N-methyl-D-aspartate (NMDA) receptors. Activation of the AMPAR-associated PI3K is required for the increased cell surface expression of AMPARs and LTP. Thus, our results strongly suggest that the AMPAR-PI3K complex may constitute a critical molecular signal responsible for AMPAR insertion at activated CA1 synapses during LTP, and consequently, this lipid kinase may serve to determine the polarity of NMDA receptor-dependent synaptic plasticity.

In the hippocampus, two distinct forms of GABAergic inhibition have been identified, phasic inhibitory postsynaptic currents that are the consequence of the vesicular release of GABA and a tonic conductance that is activated by low ambient concentrations of extracellular GABA. It is not known what accounts for the distinct properties of receptors that mediate the phasic and tonic inhibitory conductances. Moreover, the physiological role of the tonic inhibitory conductance remains uncertain because pharmacological tools that clearly distinguish tonic and phasic receptors are lacking. Here, we demonstrate that GABAA receptors that generate a tonic conductance in cultured hippocampal neurons from embryonic mice have different pharmacological properties than those in cerebellar granule neurons or pyramidal neurons in the dentate gyrus. The tonic conductance in cultured hippocampal neurons is enhanced by the benzodiazepine, midazolam, and is insensitive to the inhibitory effects of the competitive antagonist, gabazine (< or =10 microM). We also identify penicillin as an uncompetitive antagonist that selectively inhibits the synaptic but not tonic conductance. GABA was applied to hippocampal neurons to investigate the properties of synaptic and extrasynaptic receptors. GABA-evoked current was composed of two components: a rapidly desensitizing current that was blocked by penicillin and a nondesensitizing current that was insensitive to penicillin blockade. The potency of GABA was greater for the penicillin-insensitive nondesensitizing current. Single-channel studies show that the gabazine-insensitive GABAA receptors have a lower unitary conductance (12 pS) than that estimated for synaptic receptors. Thus, specialized GABAA receptors with an apparent higher affinity for GABA that do not readily desensitize mediate the persistent tonic conductance in hippocampal neurons. The receptors underlying tonic and phasic inhibitory conductances in hippocampal neurons are pharmacologically and biophysically distinct, suggesting that they serve different physiological roles.

Glycine receptors (GlyRs) are transmitter-gated channels that mediate fast inhibitory neurotransmission in the spinal cord and brain. The GlyR beta subunit contains a putative tyrosine phosphorylation site whose functional role has not been determined. To examine if protein tyrosine kinases (PTKs) regulate the function of GlyRs, we analysed whole-cell currents activated by applications of glycine to CA1 hippocampal neurons and spinal neurons. The role of a putative site for tyrosine phosphorylation at position 413 of the beta subunit was examined using site-directed mutagenesis and expression of recombinant (alpha(1)beta(Y413F)) receptors in human embryonic kidney (HEK 293) cells. Lavendustin A, an inhibitor of PTKs, depressed glycine-evoked currents (I(Gly)) in CA1 neurons and spinal neurons by 31 % and 40 %, respectively. In contrast, the intracellular application of the exogenous tyrosine kinase, cSrc, enhanced I(Gly) in CA1 neurons by 56 %. cSrc also accelerated GlyR desensitization and increased the potency of glycine 2-fold (control EC(50) = 143 microM; cSrc EC(50) = 74 microM). Exogenous cSrc, applied intracellularly, upregulated heteromeric alpha(1)beta receptors but not homomeric alpha(1) receptors. Substitution mutation of the tyrosine to phenylalanine at position beta-413 prevented this enhancement. Furthermore, a selective inhibitor of the Src family kinases, PP2, down-regulated wild-type alpha(1)beta but not alpha(1)beta(Y413F) receptors. Together, these findings indicate that GlyR function is upregulated by PTKs and this modulation is dependent on the tyrosine-413 residue of the beta subunit.

The intricate and complex interaction between different populations of neurons in the brain has imposed limits on our ability to gain detailed understanding of synaptic transmission and its integration when employing classical electrophysiological approaches. Indeed, electrical field stimulation delivered via traditional microelectrodes does not permit the targeted, precise and selective control of neuronal activity amongst a varied population of neurons and their inputs (eg, cholinergic, dopaminergic or glutamatergic neurons). Recently established optogenetic techniques overcome these limitations allowing precise control of the target neuron populations, which is essential for the elucidation of the neural substrates underlying complex animal behaviors. Indeed, by introducing light-activated channels (ie, microbial opsin genes) into specific neuronal populations, optogenetics enables non-invasive optical control of specific neurons with milliseconds precision. These approaches can readily be applied to freely behaving live animals. Recently there is increased interests in utilizing optogenetics tools to understand synaptic plasticity and learning/memory. Here, we summarize recent progress in applying optogenetics in in the study of synaptic plasticity. [ABSTRACT FROM AUTHOR]

Together, acid-sensing ion channels (ASICs) and epithelial sodium channels (ENaC) constitute the majority of voltageindependent sodium channels in mammals. ENaC is regulated by a chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR). Here we show that ASICs were reversibly inhibited by activation of GABAA receptors in murine hippocampal neurons. This inhibition of ASICs required opening of the chloride channels but occurred with both outward and inward GABAA receptor-mediated currents. Moreover, activation of the GABAA receptors modified the pharmacological features and kinetic properties of the ASIC currents, including the time course of activation, desensitization and deactivation. Modification of ASICs by open GABAA receptors was also observed in both nucleated patches and outsideout patches excised from hippocampal neurons. Interestingly, ASICs and GABAA receptors interacted to regulate synaptic plasticity in CA1 hippocampal slices. The activation of glycine receptors, which are similar to GABAA receptors, also modified ASICs in spinal neurons. We conclude that GABAA receptors and glycine receptors modify ASICs in neurons through mechanisms that require the opening of chloride channels. [ABSTRACT FROM AUTHOR]

The tyrosine kinase Pyk2 plays a unique role in intracellular signal transduction by linking Ca2+ influx to tyrosine phosphorylation, but the molecular mechanism of Pyk2 activation is unknown. We report that Pyk2 oligomerization by antibodies in vitro or overexpression of PSD-95 in PC6-3 cells induces trans-autophosphorylation of Tyr402, the first step in Pyk2 activation. In neurons, Ca2+ influx through NMDA-type glutamate receptors causes postsynaptic clustering and autophosphorylation of endogenous Pyk2 via Ca2+- and calmodulin-stimulated binding to PSD-95. Accordingly, Ca2+ influx promotes oligomerization and thereby autoactivation of Pyk2 by stimulating its interaction with PSD-95. We show that this mechanism of Pyk2 activation is critical for long-term potentiation in the hippocampus CA1 region, which is thought to underlie learning and memory. [ABSTRACT FROM AUTHOR]

Concentrations of extracellular divalent cations (Ca2+ and Mg2+) fall substantially during intensive synaptic transmission as well as during some pathophysiological conditions such as epilepsy and brain ischemia. Here we report that a synthetic serine protease inhibitor, nafamostat mesylate (NM), and several of its analogues, block recombinant TRPM7 currents expressed in HEK293T cells in inverse relationship to the concentration of extracellular divalent cations. Lowering extracellular Ca2+ and Mg2+ also evokes a divalent-sensitive non-selective cation current that is mediated by TRPM7 expression in hippocampal neurons. In cultured hippocampal neurons, NM blocked these TRPM7-mediated currents with an apparent affinity of 27 μM, as well as the paradoxical Ca2+ influx associated with lowering extracellular Ca2+. Unexpectedly, pre-exposure to NM strongly potentiated TRPM7 currents. In the presence of physiological concentrations of extracellular divalent cations, NM activates TRPM7. The stimulating effects of NM on TRPM7 currents are also inversely related to extracellular Ca2+ and Mg2+. DAPI and HSB but not netropsin, blocked and stimulated TRPM7. In contrast, mono-cationic, the metabolites of NM, p-GBA and AN, as well as protease inhibitor leupeptin and gabexate failed to substantially modulate TRPM7. NM thus provides a molecular template for the design of putative modulators of TRPM7. [ABSTRACT FROM AUTHOR]

Exposure to low Ca2+ and/or Mg2+ is tolerated by cardiac myocytes, astrocytes, and neurons, but restoration to normal divalent cation levels paradoxically causes Ca2+ overload and cell death. This phenomenon has been called the ‘Ca2+ paradox’ of ischemia-reperfusion. The mechanism by which a decrease in extracellular Ca2+ and Mg2+ is ‘detected’ and triggers subsequent cell death is unknown. Transient periods of brain ischemia are characterized by substantial decreases in extracellular Ca2+ and Mg2+ that mimic the initial condition of the Ca2+ paradox. In CA1 hippocampal neurons, lowering extracellular divalents stimulates a nonselective cation current. We show that this current resembles TRPM7 currents in several ways. Both (i) respond to transient decreases in extracellular divalents with inward currents and cell excitation, (ii) demonstrate outward rectification that depends on the presence of extracellular divalents, (iii) are inhibited by physiological concentrations of intracellular Mg2+, (iv) are enhanced by intracellular phosphatidylinositol 4,5-bisphosphate (PIP2), and (v) can be inhibited by Gaq-linked G protein-coupled receptors linked to phospholipase C β-induced hydrolysis of PIP2. Furthermore, suppression of TRPM7 expression in hippocampal neurons strongly depressed the inward currents evoked by lowering extracellular divalents. Finally, we show that activation of TRPM7 channels by lowering divalents significantly contributes to cell death. Together, the results demonstrate that TRPM7 contributes to the mechanism by which hippocampal neurons ‘detect’ reductions in extracellular divalents and provide a means by which TRPM7 contributes to neuronal death during transient brain ischemia. [ABSTRACT FROM AUTHOR]

NMDA receptor function is modulated by both G-protein-coupled receptors and receptor tyrosine kinases. In acutely isolated rat hippocampal neurons, direct activation of the platelet-derived growth factor (PDGF) receptor or transactivation of the PDGF receptor by D4 dopamine receptors inhibits NMDA-evoked currents in a phospholipase C (PLC)-dependent manner. We have investigated further the ability of D2-class dopamine receptors to modulate NMDA-evoked currents in isolated rat prefrontal cortex (PFC). We have demonstrated that, similar to isolated hippocampal neurons, the application of PDGF-BB or quinpirole to isolated PFC neurons induces a slow-onset and long-lasting inhibition of NMDA-evoked currents. However, in contrast to hippocampal neurons, the inhibition of NMDA-evoked currents by quinpirole in PFC neurons is dependent upon D2/3, rather than D4, dopamine receptors. In PFC slices, application of both PDGF-BB and quinpirole induced a phosphorylation of the PDGF receptor at the PLCγ binding and activation site, Tyr1021. The PDGF receptor kinase inhibitor, tyrphostin A9, and the D2/3 dopamine receptor antagonist, raclopride, inhibited quinpirole-induced Tyr1021 phosphorylation. These finding suggest that quinpirole treatment inhibits NMDAR signaling via PDGF receptor transactivation in both the hippocampus and the PFC, and that the effects of quinpirole in these regions are mediated by D4 and D2/3 dopamine receptors, respectively. [ABSTRACT FROM AUTHOR]

A fundamental objective of anesthesia research is to identify the receptors and brain regions that mediate the various behavioral components of the anesthetic state, including amnesia, immobility, and unconsciousness. Using complementary in vivo and in vitro approaches, we found that GABAA receptors that contain the α5 subunit (α5GABAARs) play a critical role in amnesia caused by the prototypic intravenous anesthetic etomidate. Whole-cell recordings from hippocampal pyramidal neurons showed that etomidate markedly increased a tonic inhibitory conductance generated by α5GABAARs, whereas synaptic transmission was only slightly enhanced. Long-term potentiation (LTP) of field EPSPs recorded in CA1 stratum radiatum was reduced by etomidate in wild-type (WT) but not α5 null mutant (α5-/-) mice. In addition, etomidate impaired memory performance of WT but not α5-/-mice for spatial and nonspatial hippocampal-dependent learning tasks. The brain concentration of etomidate associated with memory impairment in vivo was comparable with that which increased the tonic inhibitory conductance and blocked LTP in vitro. The α5-/-mice did not exhibit a generalized resistance to etomidate, in that the sedative-hypnotic effects measured with the rotarod, loss of righting reflex, and spontaneous motor activity were similar in WT and α5-/- mice. Deletion of the α5 subunit of the GABAARs reduced the amnestic but not the sedative-hypnotic properties of etomidate. Thus, the amnestic and sedative-hypnotic properties of etomidate can be dissociated on the basis of GABAAR subtype pharmacology. [ABSTRACT FROM AUTHOR]

Since the early 1960s it has been known that restoring extracellular Ca2+ following a period of low Ca2+ concentrations paradoxically causes an increase in intracellular Ca2+ levels that can lead to cell death. The mystery of this ‘Ca2+ paradox’ is made more intriguing by observations that lowering concentrations of extracellular Ca2+ and/or Mg2+ paradoxically enhances the entry of Ca2+ into hippocampal neurons. Until recently, the entry of Ca2+ through NMDA receptors was accepted as the major pathway leading to the excitotoxic, delayed cell death associated with the ischemic periods of stroke. Here, we discuss how several transient receptor potential (TRP) channels are likely to contribute to both the Ca2+ paradox and the delayed death of neurons following an ischemic stroke. [Copyright &y& Elsevier]

Volatile (inhaled) anesthetics cause amnesia at concentrations well below those that cause loss of consciousness and immobility; however, the underlying neuronal mechanisms are unknown. Although many anesthetics increase inhibitory GABAergic synaptic transmission, this effect occurs only at high concentrations (> 100 µM). Molecular targets for low concentrations of inhaled anesthetics have not been identified. Here, we report that a tonic inhibitory conductance in hippocampal pyramidal neurons generated by α5 subunit-containing GABAA receptors is highly sensitive to low concentrations of the volatile anesthetic isoflurane (ISO) (25 and 83.3 µM). The α5 subunit is necessary for enhancement of the tonic current by these low concentrations of isoflurane because potentiation is absent in neurons from α5-/- mice. Furthermore, ISO (25 µM) potentiated recombinant human α5β3γ2L GABAA receptors, whereas this effect was not seen α1β3γ2L GABAA with receptors. These studies suggest that an increased tonic inhibition in the hippocampus may contribute to amnestic properties of volatile anesthetics. [ABSTRACT FROM AUTHOR]

Positive modulators of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) channels reduce desensitization and alter their gating kinetics. We have discovered a novel compound nitric oxide-mimetic that similarly modulates the AMPA receptor by reducing desensitization. This, designated GT-005, belongs to the organic nitrate family that includes the nitrovasodilator nitroglycerine. In acutely isolated hippocampal neurons, GT-005 enhanced kainate (100 µM)-evoked currents with an EC[sub 50] of 1.7 ± 0.2 mM and a 176 ± 10% maximal increase in the steady-state current response. Similar results were found in cultured hippocampal neurons (EC[sub 50] of 1.3 ± 0.2 mM and a maximal 83 ± 14% increase in the steady-state current response). GT-005 reduced the desensitization of glutamate-evoked currents and slowed the onset of desensitization. This compound also increased the rate of recovery from the desensitized state. With respect to alteration of the excitatory synaptic transmission, GT-005 delayed the decay and increased the frequency of spontaneous miniature excitatory postsynaptic currents (mepsc) recorded in cultured hippocampal neurons.Key words: AMPA receptors, desensitization, organic nitrates.Les modulateurs positifs des canaux AMPA (acide α-amino-3-hydroxy-5-méthyl-4-isoxazole propionique) réduisent la désensibilisation et modifient leur cinétique d'activation. Nous avons découvert un nouveau composé mimétique du monoxyde d'azote qui module aussi le récepteur AMPA en réduisant la désensibilisation. Ce composé, appelé GT-005, appartient à la famille des nitrates organiques qui comprend le nitrovasodilatateur nitroglycérine. Dans des neurones d'hippocampe isolés de manière aiguë, GT-005 a stimulé les courants induits par le kaïnate (100 µM) avec une valeur de CE[sub 50] de 1,7 ± 0,2 mM et une augmentation de 176 ± 10 % de la réponse du courant à l'état d'équilibre. Des résultats similaires ont été obtenus dans des neurones d'hippocampe en culture (CE[sub 50] de 13 ± 0,2 mM et augmentation de 83 ± 14 % de la réponse du courant à l'état d'équilibre). GT-005 a réduit la désensibilisation des courants induits par le glutamate et ralenti le début de la désensibilisation. Ce composé a aussi augmenté le taux de rétablissement aux valeurs antérieures à l'état désensibilisé. Pour ce qui est de la modification de la transmission synaptique excitatrice, GT-005 a retardé la diminution et augmenté la fréquence des courants postsynaptiques excitateurs miniatures spontanés (cpsem) enregistrés dans les neurones hippocampiques en culture.Mots clés : récepteurs AMPA, désensibilisation, nitrates organiques.[Traduit par la Rédaction] [ABSTRACT FROM AUTHOR]

Excitotoxicity in brain ischemia triggers neuronal death and neurological disability, and yet these are not prevented by antiexcitotoxic therapy (AET) in humans. Here, we show that in neurons subjected to prolonged oxygen glucose deprivation (OGD), AET unmasks a dominant death mechanism perpetuated by a Ca2+-permeable nonselective cation conductance (IOGD). IOGD was activated by reactive oxygen/nitrogen species (ROS), and permitted neuronal Ca2+ overload and further ROS production despite AET. IOGD currents corresponded to those evoked in HEK-293 cells expressing the nonselective cation conductance TRPM7. In cortical neurons, blocking IOGD or suppressing TRPM7 expression blocked TRPM7 currents, anoxic 45Ca2+ uptake, ROS production, and anoxic death. TRPM7 suppression eliminated the need for AET to rescue anoxic neurons and permitted the survival of neurons previously destined to die from prolonged anoxia. Thus, excitotoxicity is a subset of a greater overall anoxic cell death mechanism, in which TRPM7 channels play a key role. [Copyright &y& Elsevier]