Receptor-mediated Synaptic Currents Research Articles

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

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

Molecular motor KIF17 is fundamental for memory and learning via differential support of synaptic NR2A/2B levels

Summary Kinesin superfamily motor protein 17 (KIF17) is a candidate transporter of N-methyl-D-aspartate (NMDA) receptor subunit 2B (NR2B). Disruption of the murine kif17 gene inhibits NR2B transport, accompanied by decreased transcription of nr2b , resulting in a loss of synaptic NR2B. In kif17 −/− hippocampal neurons, the NR2A level is also decreased because of accelerated ubiquitin-proteasome system-dependent degradation. Accordingly, NMDA receptor-mediated synaptic currents, early and late long-term potentiation, long-term depression, and CREB responses are attenuated in kif17 −/− neurons, concomitant with a hippocampus-dependent memory impairment in knockout mice. In wild-type neurons, CREB is activated by synaptic inputs, which increase the levels of KIF17 and NR2B. Thus, KIF17 differentially maintains the levels of NR2A and NR2B, and, when synapses are stimulated, the NR2B/KIF17 complex is upregulated on demand through CREB activity. These KIF17-based mechanisms for maintaining NR2A/2B levels could underlie multiple phases of memory processes in vivo.

Modulation of NMDA receptor function by inhibition of d-amino acid oxidase in rodent brain

Observations that N-Methyl- d-Aspartate (NMDA) antagonists produce symptoms in humans that are similar to those seen in schizophrenia have led to the current hypothesis that schizophrenia might result from NMDA receptor hypofunction. Inhibition of d-amino acid oxidase (DAAO), the enzyme responsible for degradation of d-serine, should lead to increased levels of this co-agonist at the NMDA receptor, and thereby provide a therapeutic approach to schizophrenia. We have profiled some of the preclinical biochemical, electrophysiological, and behavioral consequences of administering potent and selective inhibitors of DAAO to rodents to begin to test this hypothesis. Inhibition of DAAO activity resulted in a significant dose and time dependent increase in d-serine only in the cerebellum, although a time delay was observed between peak plasma or brain drug concentration and cerebellum d-serine response. Pharmacokinetic/pharmacodynamic (PK/PD) modeling employing a mechanism-based indirect response model was used to characterize the correlation between free brain drug concentration and d-serine accumulation. DAAO inhibitors had little or no activity in rodent models considered predictive for antipsychotic activity. The inhibitors did, however, affect cortical activity in the Mescaline-Induced Scratching model, produced a modest but significant increase in NMDA receptor-mediated synaptic currents in primary neuronal cultures from rat hippocampus, and resulted in a significant increase in evoked hippocampal theta rhythm, an in vivo electrophysiological model of hippocampal activity. These findings demonstrate that although DAAO inhibition did not cause a measurable increase in d-serine in forebrain, it did affect hippocampal and cortical activity, possibly through augmentation of NMDA receptor-mediated currents.

Proteomics, Ultrastructure, and Physiology of Hippocampal Synapses in a Fragile X Syndrome Mouse Model Reveal Presynaptic Phenotype

Fragile X syndrome (FXS), the most common form of hereditary mental retardation, is caused by a loss-of-function mutation of the Fmr1 gene, which encodes fragile X mental retardation protein (FMRP). FMRP affects dendritic protein synthesis, thereby causing synaptic abnormalities. Here, we used a quantitative proteomics approach in an FXS mouse model to reveal changes in levels of hippocampal synapse proteins. Sixteen independent pools of Fmr1 knock-out mice and wild type mice were analyzed using two sets of 8-plex iTRAQ experiments. Of 205 proteins quantified with at least three distinct peptides in both iTRAQ series, the abundance of 23 proteins differed between Fmr1 knock-out and wild type synapses with a false discovery rate (q-value) <5%. Significant differences were confirmed by quantitative immunoblotting. A group of proteins that are known to be involved in cell differentiation and neurite outgrowth was regulated; they included Basp1 and Gap43, known PKC substrates, and Cend1. Basp1 and Gap43 are predominantly expressed in growth cones and presynaptic terminals. In line with this, ultrastructural analysis in developing hippocampal FXS synapses revealed smaller active zones with corresponding postsynaptic densities and smaller pools of clustered vesicles, indicative of immature presynaptic maturation. A second group of proteins involved in synaptic vesicle release was up-regulated in the FXS mouse model. In accordance, paired-pulse and short-term facilitation were significantly affected in these hippocampal synapses. Together, the altered regulation of presynaptically expressed proteins, immature synaptic ultrastructure, and compromised short-term plasticity points to presynaptic changes underlying glutamatergic transmission in FXS at this stage of development.

Molecular Motor KIF17 Is Fundamental for Memory and Learning via Differential Support of Synaptic NR2A/2B Levels

Kinesin superfamily motor protein 17 (KIF17) is a candidate transporter of N-methyl-D-aspartate (NMDA) receptor subunit 2B (NR2B). Disruption of the murine kif17 gene inhibits NR2B transport, accompanied by decreased transcription of nr2b, resulting in a loss of synaptic NR2B. In kif17(-/-) hippocampal neurons, the NR2A level is also decreased because of accelerated ubiquitin-proteasome system-dependent degradation. Accordingly, NMDA receptor-mediated synaptic currents, early and late long-term potentiation, long-term depression, and CREB responses are attenuated in kif17(-/-) neurons, concomitant with a hippocampus-dependent memory impairment in knockout mice. In wild-type neurons, CREB is activated by synaptic inputs, which increase the levels of KIF17 and NR2B. Thus, KIF17 differentially maintains the levels of NR2A and NR2B, and, when synapses are stimulated, the NR2B/KIF17 complex is upregulated on demand through CREB activity. These KIF17-based mechanisms for maintaining NR2A/2B levels could underlie multiple phases of memory processes invivo.

Growth hormone rescues hippocampal synaptic function after sleep deprivation

Sleep is required for, and sleep loss impairs, normal hippocampal synaptic N-methyl-D-aspartate (NMDA) glutamate receptor function and expression, hippocampal NMDA receptor-dependent synaptic plasticity, and hippocampal-dependent memory function. Although sleep is essential, the signals linking sleep to hippocampal function are not known. One potential signal is growth hormone. Growth hormone is released during sleep, and its release is suppressed during sleep deprivation. If growth hormone links sleep to hippocampal function, then restoration of growth hormone during sleep deprivation should prevent adverse consequences of sleep loss. To test this hypothesis, we examined rat hippocampus for spontaneous excitatory synaptic currents in CA1 pyramidal neurons, long-term potentiation in area CA1, and NMDA receptor subunit proteins in synaptic membranes. Three days of sleep deprivation caused a significant reduction in NMDA receptor-mediated synaptic currents compared with control treatments. When rats were injected with growth hormone once per day during sleep deprivation, the loss of NMDA receptor-mediated synaptic currents was prevented. Growth hormone injections also prevented the impairment of long-term potentiation that normally follows sleep deprivation. In addition, sleep deprivation led to a selective loss of NMDA receptor 2B (NR2B) from hippocampal synaptic membranes, but normal NR2B expression was restored by growth hormone injection. Our results identify growth hormone as a critical mediator linking sleep to normal synaptic function of the hippocampus.

Input-specific synaptic plasticity in the amygdala is regulated by neuroligin-1 via postsynaptic NMDA receptors

Despite considerable evidence for a critical role of neuroligin-1 in the specification of excitatory synapses, the cellular mechanisms and physiological roles of neuroligin-1 in mature neural circuits are poorly understood. In mutant mice deficient in neuroligin-1, or adult rats in which neuroligin-1 was depleted, we have found that neuroligin-1 stabilizes the NMDA receptors residing in the postsynaptic membrane of amygdala principal neurons, which allows for a normal range of NMDA receptor-mediated synaptic transmission. We observed marked decreases in NMDA receptor-mediated synaptic currents at afferent inputs to the amygdala of neuroligin-1 knockout mice. However, the knockout mice exhibited a significant impairment in spike-timing-dependent long-term potentiation (STD-LTP) at the thalamic but not the cortical inputs to the amygdala. Subsequent electrophysiological analyses indicated that STD-LTP in the cortical pathway is largely independent of activation of postsynaptic NMDA receptors. These findings suggest that neuroligin-1 can modulate, in a pathway-specific manner, synaptic plasticity in the amygdala circuits of adult animals, likely by regulating the abundance of postsynaptic NMDA receptors.

Correlation between synaptic GABA-A receptor subunit composition and the kinetics and pharmacology of GABA-A receptor-mediated synaptic currents

Event Abstract Back to Event Correlation between synaptic GABA-A receptor subunit composition and the kinetics and pharmacology of GABA-A receptor-mediated synaptic currents Mark D. Eyre1* and Zoltán Nusser1 1 Hungarian Academy of Sciences, Laboratory of Cellular Neurophysiology, Institute of Experimental Medicine, Hungary Three different subunit types (α, β and γ) are required to compose most functional synaptic GABA-A receptors (GABA-A R), forming a stoichiometry of two α, two β and one γ subunits. Different subunit isoforms confer distinct receptor kinetics and pharmacological modulation when expressed in HEK cells. How these functional properties depend on the subunit composition in GABAergic synapses is less clear. Here we used whole-cell patch clamp recordings of miniature IPSCs in acute slices from identified neurons to address the relationship between IPSC kinetics, benzodiazepine pharmacology and synaptic subunit expression. The benzodiazepine agonist zolpidem has ~100-fold greater affinity for α1-containing GABA-A Rs compared to α2 and α3-containing ones, making it an amenable tool (at 100 nM) for studying the subunit composition of synaptic GABA-A Rs. First, we investigated cerebellar stellate cells that contain only the α1 as α subunit and main olfactory bulb (MOB) granule cells that express only the α2 as α subunit in their synapses. mIPSCs in stellate cells had an average weighted decay time constant (tw) of 4.8±0.2 ms and zolpidem slowed the decay time constant by 43.8±1.3%. In contrast, mIPSCs in granule cells were 3-fold slower (tw=14.2±0.8 ms) and showed no slowing of the decay by zolpidem (-4.9±5.5%). Next, we investigated mIPSCs in cells that express multiple synaptic α and β subunits. Deep short axon cells (dSACs) and external tufted cells (ETCs) of the MOB heterogeneously express the α1/α3 and β2/β3 subunits. Our results demonstrate that their mIPSC decay times were slower (ETCs tw=8.1±1.8 ms; dSACs tw=7.1±0.9 ms) than those of stellate cells. The zolpidem sensitivity of mIPSC decays in these cells will be compared to the immunofluorescent labeling of α1, α3, β2 and b3 subunits. Our pharmacological and kinetic results are consistent with the subunit composition of synaptic GABA-A Rs of these cells. Conference: IBRO International Workshop 2010, Pécs, Hungary, 21 Jan - 23 Jan, 2010. Presentation Type: Poster Presentation Topic: Cellular neuroscience Citation: Eyre MD and Nusser Z (2010). Correlation between synaptic GABA-A receptor subunit composition and the kinetics and pharmacology of GABA-A receptor-mediated synaptic currents. Front. Neurosci. Conference Abstract: IBRO International Workshop 2010. doi: 10.3389/conf.fnins.2010.10.00036 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 19 Apr 2010; Published Online: 19 Apr 2010. * Correspondence: Mark D Eyre, Hungarian Academy of Sciences, Laboratory of Cellular Neurophysiology, Institute of Experimental Medicine, Budapest, Hungary, eyre@koki.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Mark D Eyre Zoltán Nusser Google Mark D Eyre Zoltán Nusser Google Scholar Mark D Eyre Zoltán Nusser PubMed Mark D Eyre Zoltán Nusser Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Alpha‐synuclein overexpression in mice alters synaptic communication in the corticostriatal pathway

alpha-Synuclein (alpha-Syn) is a presynaptic protein implicated in Parkinson's disease (PD). Mice overexpressing human wildtype (WT) alpha-Syn under the Thy1 promoter show high levels of alpha-Syn in cortical and subcortical regions, exhibit progressive sensorimotor anomalies, as well as non-motor abnormalities and are considered models of pre-manifest PD as there is little evidence of early loss of dopaminergic (DA) neurons. We used whole-cell patch clamp recordings from visually identified striatal medium-sized spiny neurons (MSSNs) in slices from alpha-Syn and WT littermate control mice at 35, 90 and 300 days of age to examine corticostriatal synaptic function. MSSNs displayed significant decreases in the frequency of spontaneous excitatory postsynaptic currents (EPSCs) in alpha-Syn mice at all ages. This difference persisted in the presence of tetrodotoxin, indicating it was independent of action potentials. Stimulation thresholds for evoking EPSCs were significantly higher and responses were smaller in alpha-Syn mice. These data suggest a decrease in neurotransmitter release at the corticostriatal synapse. At 90 days the frequency of spontaneous GABA(A) receptor-mediated synaptic currents was decreased in MSSNs but increased in cortical pyramidal neurons. These observations indicate that high levels of expression of alpha-Syn alter corticostriatal synaptic function early and they provide evidence for early synaptic dysfunction in a pre-manifest model of PD. Of importance, these changes are opposite to those found in DA-depletion models, suggesting that before degeneration of DA neurons in the substantia nigra synaptic adaptations occur at the corticostriatal synapse that may initiate subtle preclinical manifestations.

Kainate receptors and signal integration by NG2 glial cells

It is well established that NG2 cells throughout the young and adult brain consistently detect the release of single vesicles filled with glutamate from nearby axons. The released neurotransmitter glutamate electrically excites NG2 cells via non-NMDA (N-methyl-D-aspartic acid) glutamate receptors but the individual contribution of AMPA and kainate receptors to neuron-NG2 cell signalling, is not well understood. Here we pharmacologically block AMPA-type glutamate receptors and investigate whether hippocampal NG2 cells also express the kainate subtype of glutamate receptors and what may be their contribution to synaptic connectivity. It has been shown previously that vesicular glutamate release does not lead to a detectable activation of kainate receptors on NG2 cells. Here we report that while bath application of 250 nM-1 muM kainate does not have a major effect on NG2 cells it consistently induces a small and persistent depolarising current. This current was not mimicked by ATPA, suggesting that this current is carried by non-GluR5 containing kainate receptors. In addition to this inward current, nanomolar concentrations of kainate also produced a dramatic increase in the frequency of spontaneous GABA-A receptor-mediated synaptic currents (IPSCs) in NG2 cells. This increase in spontaneous IPSC frequency was even more pronounced on application of the GluR5-specific agonist ATPA (approximately 15-fold increase in frequency). In contrast, mono-synaptic stimulated IPSCs recorded in NG2 cells were unaffected by kainate receptor activation. Those and further experiments show that the occurrence of the high frequency of IPSCs is due to action potential firing of hippocampal interneurons caused by activation of GluR5 receptors on the somatodendritic membrane of the interneurons. Our data suggest that hippocampal kainate receptors are not only important for communication between neurons but may also play a dual and subtype-specific role for neuron-glia signalling: Firstly, extra-synaptic non-GluR5 kainate receptors in the membrane of NG2 cells are ideally suited to instruct NG2 cells on the population activity of local excitatory neurons via ambient glutamate. Secondly, based on the known importance of GluR5 receptors on hippocampal interneurons for the generation of network rhythms and based on our finding that these interneurons heavily project onto NG2 cells, it appears that synaptic activation of interneuronal GluR5 receptors triggers signalling to NG2 cells which transmits the phase and frequency of ongoing network oscillations in the developing hippocampus.

Type A GABA-Receptor-Dependent Synaptic Transmission Sculpts Dendritic Arbor Structure inXenopusTadpolesIn Vivo

The emergence of dendritic arbor structure in vivo depends on synaptic inputs. We tested whether inhibitory GABAergic synaptic transmission regulates Xenopus optic tectal cell dendritic arbor development in vivo by expressing a peptide corresponding to an intracellular loop (ICL) of the gamma2 subunit of type A GABA receptors (GABA(A)R), which is required to anchor GABA(A) receptors to the postsynaptic scaffold. Enhanced green fluorescent protein (EGFP)-tagged ICL (EGFP-ICL) was distributed in a punctate pattern at putative inhibitory synapses, identified by vesicular GABA transporter immunoreactive puncta. ICL expression completely blocked GABA(A)R-mediated transmission in 36% of transfected neurons and significantly reduced GABA(A)R-mediated synaptic currents relative to AMPA receptor-mediated synaptic currents in the remaining transfected neurons without altering release probability or neuronal excitability. Further analysis of ICL-expressing neurons with residual GABA(A)R-mediated inputs showed that the capacity of benzodiazepine to enhance GABAergic synaptic responses was reduced in ICL-expressing neurons, indicating that they were likely depleted of gamma2 subunit-containing GABA(A)R. Neurons expressing a mutant form of ICL were comparable to controls. In vivo time-lapse images showed that ICL-expressing neurons have more sparsely branched dendritic arbors, which expand over larger neuropil areas than EGFP-expressing control neurons. Analysis of branch dynamics indicated that ICL expression affected arbor growth by reducing rates of branch addition. Furthermore, we found that decreasing GABAergic synaptic transmission with ICL expression blocked visual experience dependent dendritic arbor structural plasticity. Our findings establish an essential role for inhibitory GABAergic synaptic transmission in the regulation of dendritic structural plasticity in Xenopus in vivo.

Substance P and cocaine employ convergent mechanisms to depress excitatory synaptic transmission in the rat nucleus accumbens in vitro

Substance P (SP) has been reported to produce effects on excitatory synaptic transmission in the nucleus accumbens (NAc) that are similar to those induced by cocaine. To address the question of whether SP serves as an endogenous mediator producing cocaine-like effects that are known to be D1-receptor-mediated, we tested the hypothesis that the effects of SP and cocaine on excitatory postsynaptic currents (EPSCs) in the NAc occlude one another. We report here that SP and SP(5-11) actions occlude the effect of cocaine and vice versa. SP, SP(5-11) and cocaine all depressed evoked, non-N-methyl-D-aspartate (NMDA) receptor-mediated synaptic currents in a concentration-dependent manner, with EC50 values of 0.12, 0.17 and 8.3 microm, respectively. Although cocaine was the least potent, it was most efficacious. SP, SP(5-11) and cocaine all suppressed isolated NMDA receptor-mediated evoked EPSCs. SP(5-11) (1 microm)-induced EPSC depression was blocked by the neurokinin-1 antagonist L732138 and by the D1-like receptor antagonist SCH23390. Pretreatment of slices with cocaine (30 microm) depressed the EPSC by 39.1% +/- 4.8%. Application of SP or SP(5-11) (1 microm) at the peak of the cocaine depressive effect on the EPSC did not produce any additional diminution of the response (5.7% +/- 2.8%). In the reverse experiments, in which either SP or SP(5-11) was applied first, subsequent application of cocaine at the peak of the peptide's effect (30.3% +/- 2.3%) produced a further but smaller depression (15.5% +/- 3.6%) of the remaining EPSC. These data indicate that cocaine and SP produce similar effects on excitatory synaptic transmission in the NAc, and that their actions occlude one another. This suggests that SP may act like cocaine in its absence, and may be an endogenous trigger for the reward and behaviors associated with cocaine.

D1/D5Modulation of Synaptic NMDA Receptor Currents

Converging evidence suggests that salience-associated modulation of behavior is mediated by the release of monoamines and that monoaminergic activation of D(1)/D(5) receptors is required for normal hippocampal-dependent learning and memory. However, it is not understood how D(1)/D(5) modulation of hippocampal circuits can affect salience-associated learning and memory. We have observed in CA1 pyramidal neurons that D(1)/D(5) receptor activation elicits a bidirectional long-term plasticity of NMDA receptor-mediated synaptic currents with the polarity of plasticity determined by NMDA receptor, NR2A/B subunit composition. This plasticity results in a decrease in the NR2A/NR2B ratio of subunit composition. Synaptic responses mediated by NMDA receptors that include NR2B subunits are potentiated by D(1)/D(5) receptor activation, whereas responses mediated by NMDA receptors that include NR2A subunits are depressed. Furthermore, these bidirectional, subunit-specific effects are mediated by distinctive intracellular signaling mechanisms. Because there is a predominance of NMDA receptors composed of NR2A subunits observed in entorhinal-CA1 inputs and a predominance of NMDA receptors composed of NR2B subunits in CA3-CA1 synapses, potentiation of synaptic NMDA currents predominates in the proximal CA3-CA1 synapses, whereas depression of synaptic NMDA currents predominates in the distal entorhinal-CA1 synapses. Finally, all of these effects are reproduced by the release of endogenous monoamines through activation of D(1)/D(5) receptors. Thus, endogenous D(1)/D(5) activation can (1) decrease the NR2A/NR2B ratio of NMDA receptor subunit composition at glutamatergic synapses, a rejuvenation to a composition similar to developmentally immature synapses, and, (2) in CA1, bias NMDA receptor responsiveness toward the more highly processed trisynaptic CA3-CA1 circuit and away from the direct entorhinal-CA1 input.

Co-expression of Argonaute2 enhances short hairpin RNA-induced RNA interference in Xenopus CNS neurons in vivo

RNA interference (RNAi) is an evolutionarily conserved mechanism for sequence-specific gene silencing. Recent advances in our understanding of RNAi machinery make it possible to reduce protein expression by introducing short hairpin RNA (shRNA) into cells of many systems, however, the efficacy of RNAi-mediated protein knockdown can be quite variable, especially in intact animals, and this limits its application. We built adaptable molecular tools, pSilencer (pSi) and pReporter (pRe) constructs, to evaluate the impact of different promoters, shRNA structures and overexpression of Ago2, the key enzyme in the RNA-induced silencing complex (RISC), on the efficiency of RNAi. The magnitude of RNAi knockdown was evaluated in cultured cells and intact animals by comparing fluorescence intensity levels of GFP, the RNAi target, relative to mCherry, which was not targeted. Co-expression of human Ago2 with shRNA significantly enhanced efficiency of GFP knockdown in cell lines and in neurons of intact Xenopus tadpoles. Human H1- and U6-promotors alone or the U6-promotor with an enhancer element were equally effective at driving GFP knockdown. shRNA derived from the microRNA-30 design (shRNAmir30) enhanced the efficiency of GFP knockdown. Expressing pSi containing Ago2 with shRNA increased knockdown efficiency of an endogenous neuronal protein, the GluR2 subunit of the AMPA receptor, functionally accessed by recording AMPA receptor-mediated spontaneous synaptic currents in Xenopus CNS neurons. Our data suggest that co-expression of Ago2 and shRNA is a simple method to enhance RNAi in intact animals. While morpholino antisense knockdown is effective in Xenopus and Zebrafish, a principle advantage of the RNAi method is the possibility of spatial and temporal control of protein knockdown by use of cell type specific and regulatable pol II promoters to drive shRNA and Ago2. This should extend the application of RNAi to study gene function of intact brain circuits.

Co-expression of Argonaute2 enhances short hairpin RNA-induced RNA interference in Xenopus CNS neurons in vivo

RNA interference (RNAi) is an evolutionarily conserved mechanism for sequence-specific gene silencing. Recent advances in our understanding of RNAi machinery make it possible to reduce protein expression by introducing short hairpin RNA (shRNA) into cells of many systems, however, the efficacy of RNAi-mediated protein knockdown can be quite variable, especially in intact animals, and this limits its application. We built adaptable molecular tools, pSilencer (pSi) and pReporter (pRe) constructs, to evaluate the impact of different promoters, shRNA structures and overexpression of Ago2, the key enzyme in the RNA-induced silencing complex, on the efficiency of RNAi. The magnitude of RNAi knockdown was evaluated in cultured cells and intact animals by comparing fluorescence intensity levels of GFP, the RNAi target, relative to mCherry, which was not targeted. Co-expression of human Ago2 with shRNA significantly enhanced efficiency of GFP knockdown in cell lines and in neurons of intact Xenopus tadpoles. Human H1- and U6-promotors alone or the U6-promotor with an enhancer element were equally effective at driving GFP knockdown. shRNA derived from the microRNA-30 design (shRNAmir30) enhanced the efficiency of GFP knockdown. Expressing pSi containing Ago2 with shRNA increased knockdown efficiency of an endogenous neuronal protein, the GluR2 subunit of the AMPA receptor, functionally accessed by recording AMPA receptor-mediated spontaneous synaptic currents in Xenopus CNS neurons. Our data suggest that co-expression of Ago2 and shRNA is a simple method to enhance RNAi in intact animals. While morpholino antisense knockdown is effective in Xenopus and Zebrafish, a principle advantage of the RNAi method is the possibility of spatial and temporal control of protein knockdown by use of cell type specific and regulatable pol II promoters to drive shRNA and Ago2. This should extend the application of RNAi to study gene function of intact brain circuits.

Bi‐directional modulation of fast inhibitory synaptic transmission by leptin

The hormone leptin has widespread actions in the CNS. Indeed, leptin markedly influences hippocampal excitatory synaptic transmission and synaptic plasticity. However, the effects of leptin on fast inhibitory synaptic transmission in the hippocampus have not been evaluated. Here, we show that leptin modulates GABA(A) receptor-mediated synaptic transmission onto hippocampal CA1 pyramidal cells. Leptin promotes a rapid and reversible increase in the amplitude of evoked GABA(A) receptor-mediated inhibitory synaptic currents (IPSCs); an effect that was paralleled by increases in the frequency and amplitude of miniature IPSCs, but with no change in paired pulse ratio or coefficient of variation, suggesting a post-synaptic expression mechanism. Following washout of leptin, a persistent depression (inhibitory long-lasting depression) of evoked IPSCs was observed. Whole-cell dialysis or bath application of inhibitors of phosphoinositide 3 (PI 3)-kinase or Akt prevented leptin-induced enhancement of IPSCs indicating involvement of a post-synaptic PI 3-kinase/Akt-dependent pathway. In contrast, blockade of PI 3-kinase or Akt activity failed to alter the ability of leptin to induce inhibitory long-lasting depression, suggesting that this process is independent of PI 3-kinase/Akt. In conclusion these data indicate that the hormone leptin bi-directionally modulates GABA(A) receptor-mediated synaptic transmission in the hippocampus. These findings have important implications for the role of this hormone in regulating hippocampal pyramidal neuron excitability.

A NMDA receptor glycine site partial agonist, GLYX-13, simultaneously enhances LTP and reduces LTD at Schaffer collateral–CA1 synapses in hippocampus

N-methyl- d-aspartate glutamate receptors (NMDARs) are a key route for Ca 2+ influx into neurons important to both activity-dependent synaptic plasticity and, when uncontrolled, triggering events that cause neuronal degeneration and death. Among regulatory binding sites on the NMDAR complex is a glycine binding site, distinct from the glutamate binding site, which must be co-activated for NMDAR channel opening. We developed a novel glycine site partial agonist, GLYX-13, which is both nootropic and neuroprotective in vivo. Here, we assessed the effects of GLYX-13 on long-term synaptic plasticity and NMDAR transmission at Schaffer collateral–CA1 synapses in hippocampal slices in vitro. GLYX-13 simultaneously enhanced the magnitude of long-term potentiation (LTP) of synaptic transmission, while reducing long-term depression (LTD). GLYX-13 reduced NMDA receptor-mediated synaptic currents in CA1 pyramidal neurons evoked by low frequency Schaffer collateral stimulation, but enhanced NMDAR currents during high frequency bursts of activity, and these actions were occluded by a saturating concentration of the glycine site agonist d-serine. Direct two-photon imaging of Schaffer collateral burst-evoked increases in [Ca 2+] in individual dendritic spines revealed that GLYX-13 selectively enhanced burst-induced NMDAR-dependent spine Ca 2+ influx. Examining the rate of MK-801 block of synaptic versus extrasynaptic NMDAR-gated channels revealed that GLYX-13 selectively enhanced activation of burst-driven extrasynaptic NMDARs, with an action that was blocked by the NR2B-selective NMDAR antagonist ifenprodil. Our data suggest that GLYX-13 may have unique therapeutic potential as a learning and memory enhancer because of its ability to simultaneously enhance LTP and suppress LTD.

Chronic benzodiazepine-induced reduction in GABA A receptor-mediated synaptic currents in hippocampal CA1 pyramidal neurons prevented by prior nimodipine injection

One week oral flurazepam (FZP) administration in rats results in reduced GABA A receptor-mediated synaptic transmission in CA1 pyramidal neurons associated with benzodiazepine tolerance in vivo and in vitro. Since voltage-gated calcium channel (VGCC) current density is enhanced twofold during chronic FZP treatment, the role of L-type VGCCs in regulating benzodiazepine-induced changes in CA1 neuron GABA A receptor-mediated function was evaluated. Nimodipine (10 mg/kg, i.p.) or vehicle (0.5% Tween 80, 2 ml/kg) was injected 1 day after ending FZP treatment and 24 h prior to hippocampal slice preparation for measurement of mIPSC characteristics and in vitro tolerance to zolpidem. The reduction in GABA A receptor-mediated mIPSC amplitude and estimated unitary channel conductance measured 2 days after drug removal was no longer observed following prior nimodipine injection. However, the single nimodipine injection failed to prevent in vitro tolerance to zolpidem's ability to prolong mIPSC decay in FZP-treated neurons, suggesting multiple mechanisms may be involved in regulating GABA A receptor-mediated synaptic transmission following chronic FZP administration. As reported previously in recombinant receptors, nimodipine inhibited synaptic GABA A receptor currents only at high concentrations (>30 μM), significantly greater than attained in vivo (1 μM) 45 min after a single antagonist injection. Thus, the effects of nimodipine were unlikely to be related to direct effects on GABA A receptors. As with nimodipine injection, buffering intracellular free [Ca 2+] with BAPTA similarly prevented the effects on GABA A receptor-mediated synaptic transmission, suggesting intracellular Ca 2+ homeostasis is important to maintain GABA A receptor function. The findings further support a role for activation of L-type VGCCs, and perhaps other Ca 2+-mediated signaling pathways, in the modulation of GABA A receptor synaptic function following chronic benzodiazepine administration, independent of modulation of the allosteric interactions between benzodiazepine and GABA binding sites.

Modulation of AMPA receptor‐mediated ion current by pituitary adenylate cyclase‐activating polypeptide (PACAP) in CA1 pyramidal neurons from rat hippocampus

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neurotrophic and neuromodulatory peptide, was recently shown to enhance NMDA receptor-mediated currents in the hippocampus (Macdonald, et al. 2005. J Neurosci 25:11374-11384). To check if PACAP might also modulate AMPA receptor function, we tested its effects on AMPA receptor-mediated synaptic currents on CA1 pyramidal neurons, using the patch clamp technique on hippocampal slices. In the presence of the NMDA antagonist D-AP5, PACAP (10 nM) reduced the amplitude of excitatory postsynaptic currents (EPSCs) evoked in CA1 pyramidal neurons by stimulation of Schaffer collaterals. Following a paired-pulse stimulation protocol, the paired-pulse ratio was unaffected in most neurons, suggesting that the AMPA-mediated EPSC was modulated by PACAP mainly at a postsynaptic level. PACAP also modulated the currents induced on CA1 pyramidal neurons by applications of either glutamate or AMPA. The effects of PACAP were dose-dependent: at a 0.5 nM dose, PACAP increased AMPA-mediated current; such effect was blocked by PACAP 6-38, a selective antagonist of PAC1 receptors. The enhancement of AMPA-mediated current by PACAP 0.5 nM was abolished when cAMPS-Rp, a PKA inhibitor, was added to the intracellular solution. At a 10 nM concentration, PACAP reduced AMPA-mediated current; such effect was not blocked by PACAP 6-38. The inhibitory effect of 10 nM PACAP was mimicked by Bay 55-9837 (a selective agonist of VPAC2 receptors), persisted in the presence of intracellular BAPTA and was abolished by intracellular cAMPS-Rp. Stimulation-evoked EPSCs in CA1 neurons were significantly reduced following application of the PAC1 antagonist PACAP 6-38; this result indicates that PAC1 receptors in the CA1 region are tonically activated by endogenous PACAP and enhance CA3-CA1 synaptic transmission. Our results show that PACAP differentially modulates AMPA receptor-mediated current in CA1 pyramidal neurons by activation of PAC1 and VPAC2 receptors, both involving the cAMP/PKA pathway; the functional significance will be discussed in light of the multiple effects exerted by PACAP on the CA3-CA1 synapse at different levels.

Plasmin Potentiates Synaptic N-Methyl-D-aspartate Receptor Function in Hippocampal Neurons through Activation of Protease-activated Receptor-1

Protease-activated receptor-1 (PAR1) is activated by a number of serine proteases, including plasmin. Both PAR1 and plasminogen, the precursor of plasmin, are expressed in the central nervous system. In this study we examined the effects of plasmin in astrocyte and neuronal cultures as well as in hippocampal slices. We find that plasmin evokes an increase in both phosphoinositide hydrolysis (EC(50) 64 nm) and Fura-2/AM fluorescence (195 +/- 6.7% above base line, EC(50) 65 nm) in cortical cultured murine astrocytes. Plasmin also activates extracellular signal-regulated kinase (ERK1/2) within cultured astrocytes. The plasmin-induced rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) and the increase in phospho-ERK1/2 levels were diminished in PAR1(-/-) astrocytes and were blocked by 1 microm BMS-200261, a selective PAR1 antagonist. However, plasmin had no detectable effect on ERK1/2 or [Ca(2+)](i) signaling in primary cultured hippocampal neurons or in CA1 pyramidal cells in hippocampal slices. Plasmin (100-200 nm) application potentiated the N-methyl-D-aspartate (NMDA) receptor-dependent component of miniature excitatory postsynaptic currents recorded from CA1 pyramidal neurons but had no effect on alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate- or gamma-aminobutyric acid receptor-mediated synaptic currents. Plasmin also increased NMDA-induced whole cell receptor currents recorded from CA1 pyramidal cells (2.5 +/- 0.3-fold potentiation over control). This effect was blocked by BMS-200261 (1 microm; 1.02 +/- 0.09-fold potentiation over control). These data suggest that plasmin may serve as an endogenous PAR1 activator that can increase [Ca(2+)](i) in astrocytes and potentiate NMDA receptor synaptic currents in CA1 pyramidal neurons.