Autism-Misregulated eIF4G Microexons Control Synaptic Translation and Higher Order Cognitive Functions.

Microexons represent the most highly conserved class of alternative splicing, yet their functions are poorly understood. Here, we focus on closely related neuronal microexons overlapping prion-like domains in the translation initiation factors, eIF4G1 and eIF4G3, the splicing of which is activity dependent and frequently disrupted in autism. CRISPR-Cas9 deletion of these microexons selectively upregulates synaptic proteins that control neuronal activity and plasticity and further triggers a gene expression program mirroring that of activated neurons. Mice lacking the Eif4g1 microexon display social behavior, learning, and memory deficits, accompanied by altered hippocampal synaptic plasticity. We provide evidence that the eIF4G microexons function as a translational brake by causing ribosome stalling, through their propensity to promote the coalescence of cytoplasmic granule components associated with translation repression, including the fragile X mental retardation protein FMRP. The results thus reveal an autism-disrupted mechanism by which alternative splicing specializes neuronal translation to control higher order cognitive functioning.

NMDAR-dependent Argonaute 2 phosphorylation regulates miRNA activity and dendritic spine plasticity.

MicroRNAs (miRNAs) repress translation of target mRNAs by associating with Argonaute (Ago) proteins to form the RNA-induced silencing complex (RISC), underpinning a powerful mechanism for fine-tuning protein expression. Specific miRNAs are required for NMDA receptor (NMDAR)-dependent synaptic plasticity by modulating the translation of proteins involved in dendritic spine morphogenesis or synaptic transmission. However, it is unknown how NMDAR stimulation stimulates RISC activity to rapidly repress translation of synaptic proteins. We show that NMDAR stimulation transiently increases Akt-dependent phosphorylation of Ago2 at S387, which causes an increase in binding to GW182 and a rapid increase in translational repression of LIMK1 via miR-134. Furthermore, NMDAR-dependent down-regulation of endogenous LIMK1 translation in dendrites and dendritic spine shrinkage requires phospho-regulation of Ago2 at S387. AMPAR trafficking and hippocampal LTD do not involve S387 phosphorylation, defining this mechanism as a specific pathway for structural plasticity. This work defines a novel mechanism for the rapid transduction of NMDAR stimulation into miRNA-mediated translational repression to control dendritic spine morphology.

GSK-3ß regulates the synaptic expression of NMDA receptors via phosphorylation of phosphatidylinositol 4 kinase type IIα.

Deregulation of GSK-3ß is strongly implicated in a variety of serious brain conditions, such as Alzheimer disease, bipolar disorder and schizophrenia. To understand how GSK-3ß becomes dysregulated in these conditions, it is important to understand its physiological functions in the central nervous system. In this context, GSK-3ß plays a role in the induction of NMDA receptor-dependent long-term depression (LTD) and several substrates for GSK-3ß have been identified in this form of synaptic plasticity, including KLC-2, PSD-95 and tau. Stabilization of NMDA receptors at synapses has also been shown to involve GSK-3ß, but the substrates involved are currently unknown. Recent work has identified phosphatidylinositol 4 kinase type IIα (PI4KIIα) as a neuronal GSK-3ß substrate that can potentially regulate the surface expression of AMPA receptors. In the present study, we investigated the synaptic role of PI4KIIα in organotypic rat hippocampal slices. We found that knockdown of PI4KIIα has no effect on synaptic AMPA receptor-mediated synaptic transmission but substantially reduces NMDA receptor-mediated synaptic transmission. Furthermore, the ability of the selective GSK-3 inhibitor, CT99021, to reduce the amplitude of NMDA receptor-mediated currents was occluded in shRNA-PI4KIIα transfected neurons. The effects of knocking down PI4KIIα were fully rescued by a shRNA-resistant wild-type construct, but not by a mutant construct that cannot be phosphorylated by GSK-3ß. These data suggest that GSK-3ß phosphorylates PI4KIIα to stabilize NMDA receptors at the synapse.

Synaptic plasticity in the anterior cingulate cortex in acute and chronic pain.

The anterior cingulate cortex (ACC) is activated in both acute and chronic pain. In this Review, we discuss increasing evidence from rodent studies that ACC activation contributes to chronic pain states and describe several forms of synaptic plasticity that may underlie this effect. In particular, one form of long-term potentiation (LTP) in the ACC, which is triggered by the activation of NMDA receptors and expressed by an increase in AMPA-receptor function, sustains the affective component of the pain state. Another form of LTP in the ACC, which is triggered by the activation of kainate receptors and expressed by an increase in glutamate release, may contribute to pain-related anxiety.

Synthesis and pharmacological characterisation of arctigenin analogues as antagonists of AMPA and kainate receptors.

(-)-Arctigenin and a series of new analogues have been synthesised and then tested for their potential as AMPA and kainate receptor antagonists of human homomeric GluA1 and GluK2 receptors expressed in HEK293 cells using a Ca2+ influx assay. In general, these compounds showed antagonist activity at both receptors with greater activity evident at AMPARs. Schild analysis indicates that a spirocyclic analogue 6c acts as a non-competitive antagonist. Molecular docking studies in which 6c was docked into the X-ray crystal structure of the GluA2 tetramer suggest that (-)-arctigenin and its analogues bind in the transmembrane domain in a similar manner to the known AMPA receptor non-competitive antagonists GYKI53655 and the antiepileptic drug perampanel. The arctigenin derivatives described herein may serve as novel leads for the development of drugs for the treatment of epilepsy.

Calcium-Permeable AMPA Receptors Mediate the Induction of the Protein Kinase A-Dependent Component of Long-Term Potentiation in the Hippocampus.

Two forms of NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) at hippocampal CA1 synapses can be distinguished based on their sensitivity to inhibitors of protein kinase A (PKA). The PKA-dependent form requires multiple episodes of high-frequency stimulation (HFS) or theta burst stimuli (TBS) with a spacing between episodes in the order of minutes. To investigate the mechanism by which spaced episodes induce the PKA-dependent form of LTP, we have compared, in interleaved experiments, spaced (s) and compressed (c) TBS protocols in the rat CA1 synapses. We find that LTP induced by sTBS, but not that induced by cTBS, involves the insertion of calcium-permeable (CP) AMPARs, as assessed using pharmacological and electrophysiological criteria. Furthermore, a single TBS when paired with rolipram [4-(3-(cyclopentyloxy)-4-methoxyphenyl)pyrrolidin-2-one], to activate PKA, generates an LTP that also involves the insertion of CP-AMPARs. These data demonstrate that the involvement of CP-AMPARs in LTP is critically determined by the timing of the induction trigger and is associated specifically with the PKA-dependent form of LTP. SIGNIFICANCE STATEMENT: Long-term potentiation is a family of synaptic mechanisms that are believed to be important for learning and memory. Two of the most extensively studied forms are triggered by the synaptic activation of NMDA receptors and expressed by changes in AMPA receptor function. They can be distinguished on the basis of their requirement for activation of a protein kinase, PKA. We show that the PKA-dependent form also involves the transient insertion of calcium-permeable AMPA receptors. These results have implications for relating synaptic plasticity to learning and memory and suggest a specific linkage between PKA activation and the rapid synaptic insertion of calcium-permeable AMPA receptors during long-term potentiation.

Hippocampal metabotropic glutamate receptor long-term depression in health and disease: focus on mitogen-activated protein kinase pathways.

Group I metabotropic glutamate receptor (mGluR) dependent long-term depression (LTD) is a major form of synaptic plasticity underlying learning and memory. The molecular mechanisms involved in mGluR-LTD have been investigated intensively for the last two decades. In this 60th anniversary special issue article, we review the recent advances in determining the mechanisms that regulate the induction, transduction and expression of mGluR-LTD in the hippocampus, with a focus on the mitogen-activated protein kinase (MAPK) pathways. In particular we discuss the requirement of p38 MAPK and extracellular signal-regulated kinase 1/2 (ERK 1/2) activation. The recent advances in understanding the signaling cascades regulating mGluR-LTD are then related to the cognitive impairments observed in neurological disorders, such as fragile X syndrome and Alzheimer's disease. mGluR-LTD is a form of synaptic plasticity that impacts on memory formation. In the hippocampus mitogen-activated protein kinases (MAPKs) have been found to be important in mGluR-LTD. In this 60th anniversary special issue article, we review the independent and complementary roles of two classes of MAPK, p38 and ERK1/2 and link this to the aberrant mGluR-LTD that has an important role in diseases. This article is part of the 60th Anniversary special issue.

An interchangeable role for kainate and metabotropic glutamate receptors in the induction of rat hippocampal mossy fiber long-term potentiation in vivo.

The roles of both kainate receptors (KARs) and metabotropic glutamate receptors (mGluRs) in mossy fiber long-term potentiation (MF-LTP) have been extensively studied in hippocampal brain slices, but the findings are controversial. In this study, we have addressed the roles of both mGluRs and KARs in MF-LTP in anesthetized rats. We found that MF-LTP could be induced in the presence of either GluK1-selective KAR antagonists or group I mGluR antagonists. However, LTP was inhibited when the group I mGluRs and the GluK1-KARs were simultaneously inhibited. Either mGlu1 or mGlu5 receptor activation is sufficient to induce this form of LTP as selective inhibition of either subtype alone, together with the inhibition of KARs, did not inhibit MF-LTP. These data suggest that mGlu1 receptors, mGlu5 receptors, and GluK1-KARs are all engaged during high-frequency stimulation, and that the activation of any one of these receptors alone is sufficient for the induction of MF-LTP in vivo.

Long-term depression in the CNS.

Long-term depression (LTD) in the CNS has been the subject of intense investigation as a process that may be involved in learning and memory and in various pathological conditions. Several mechanistically distinct forms of this type of synaptic plasticity have been identified and their molecular mechanisms are starting to be unravelled. Most studies have focused on forms of LTD that are triggered by synaptic activation of either NMDARs (N-methyl-D-aspartate receptors) or metabotropic glutamate receptors (mGluRs). Converging evidence supports a crucial role of LTD in some types of learning and memory and in situations in which cognitive demands require a flexible response. In addition, LTD may underlie the cognitive effects of acute stress, the addictive potential of some drugs of abuse and the elimination of synapses in neurodegenerative diseases.

G protein-coupled receptor kinase 2 and group I metabotropic glutamate receptors mediate inflammation-induced sensitization to excitotoxic neurodegeneration.

OBJECTIVE: The concept of inflammation-induced sensitization is emerging in the field of perinatal brain injury, stroke, Alzheimer disease, and multiple sclerosis. However, mechanisms underpinning this process remain unidentified. METHODS: We combined in vivo systemic lipopolysaccharide-induced or interleukin (IL)-1ß-induced sensitization of neonatal and adult rodent cortical neurons to excitotoxic neurodegeneration with in vitro IL-1ß sensitization of human and rodent neurons to excitotoxic neurodegeneration. Within these inflammation-induced sensitization models, we assessed metabotropic glutamate receptors (mGluR) signaling and regulation. RESULTS: We demonstrate for the first time that group I mGluRs mediate inflammation-induced sensitization to neuronal excitotoxicity in neonatal and adult neurons across species. Inflammation-induced G protein-coupled receptor kinase 2 (GRK2) downregulation and genetic deletion of GRK2 mimicked the sensitizing effect of inflammation on excitotoxic neurodegeneration. Thus, we identify GRK2 as a potential molecular link between inflammation and mGluR-mediated sensitization. INTERPRETATION: Collectively, our findings indicate that inflammation-induced sensitization is universal across species and ages and that group I mGluRs and GRK2 represent new avenues for neuroprotection in perinatal and adult neurological disorders.

Acute stress causes rapid synaptic insertion of Ca2+ -permeable AMPA receptors to facilitate long-term potentiation in the hippocampus.

The neuroendocrine response to episodes of acute stress is crucial for survival whereas the prolonged response to chronic stress can be detrimental. Learning and memory are particularly susceptible to stress with cognitive deficits being well characterized consequences of chronic stress. Although there is good evidence that acute stress can enhance cognitive performance, the mechanism(s) for this are unclear. We find that hippocampal slices, either prepared from rats following 30 min restraint stress or directly exposed to glucocorticoids, exhibit an N-methyl-d-aspartic acid receptor-independent form of long-term potentiation. We demonstrate that the mechanism involves an NMDA receptor and PKA-dependent insertion of Ca2+ -permeable AMPA receptors into synapses. These then trigger the additional NMDA receptor-independent form of LTP during high frequency stimulation.

Memantine: Updating a rare success story in pro-cognitive therapeutics.

The great potential for NMDA receptor modulators as druggable targets in neurodegenerative disorders has been met with limited success. Considered one of the rare exceptions, memantine has consistently demonstrated restorative and prophylactic properties in many AD models. In clinical trials memantine slows the decline in cognitive performance associated with AD. Here, we provide an overview of the basic properties including pharmacological targets, toxicology and cellular effects of memantine. Evidence demonstrating reductions in molecular, physiological and behavioural indices of AD-like impairments associated with memantine treatment are also discussed. This represents both an extension and homage to Dr. Chris Parson's considerable contributions to our fundamental understanding of a success story in the AD treatment landscape.

Sex-dependence of synaptic depression induced by activation of metabotropic glutamate receptors in rat hippocampus.

The modulation of synaptic efficacy by group I metabotropic glutamate receptors is dysregulated in several neurodevelopmental and neurodegenerative disorders impacting cognitive function. The progression and severity of these and other disorders are affected by biological sex, and differences in metabotropic glutamate receptor signalling have been implicated in this effect. In this study, we have examined whether there are any sex-dependent differences in a form of long-term depression of synaptic responses that is triggered by application of the group I metabotropic glutamate receptor agonist 3,5-dihydroxyphenylglycine (DHPG). We studied DHPG-induced long-term depression at the Schaffer collateral-commissural pathway in area CA1 of hippocampal slices prepared from three separate age groups of Sprague Dawley rats. In both juvenile (2-week-old) and young adult (3-month-old) rats, there were no differences between sexes in the magnitude of long-term depression. However, in older adult (>1-year-old) rats, DHPG-induced long-term depression was greater in males. In contrast, there were no differences between sexes with respect to basal synaptic transmission or paired-pulse facilitation in any age group. The specific enhancement of metabotropic glutamate receptor-dependent long-term depression in older adult males, but not females, reinforces the importance of considering sex as a factor in the study and treatment of brain disorders.

The role of calcium stores in long-term potentiation and synaptic tagging and capture in mouse hippocampus.

The roles of Ca2+-induced calcium release in synaptic plasticity and metaplasticity are poorly understood. The present study has addressed the role of intracellular Ca2+ stores in long-term potentiation (LTP) and a form of heterosynaptic metaplasticity known as synaptic tagging and capture (STC) at CA1 synapses in mouse hippocampal slices. The effects of two compounds, ryanodine and cyclopiazonic acid (CPA), were examined on LTP induced by three distinct induction protocols: weak (w), compressed (c) and spaced (s) theta-burst stimulation (TBS). These compounds did not significantly affect LTP induced by the wTBS (one episode of TBS; 25 stimuli) or cTBS (three such episodes with a 10 s inter-episode interval (IEI); 75 stimuli) but substantially inhibited LTP induced by a sTBS (10 min IEI; 75 stimuli). Ryanodine and CPA also prevented a small heterosynaptic potentiation that was observed with the sTBS protocol. Interestingly, these compounds also prevented STC when present during either the sTBS or the subsequent wTBS, applied to an independent input. All of these effects of ryanodine and CPA were similar to that of a calcium-permeable AMPA receptor blocker. In conclusion, Ca2+ stores provide one way in which signals are propagated between synaptic inputs and, by virtue of their role in STC, may be involved in associative long-term memories. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Adiponectin rescues synaptic plasticity in the dentate gyrus of a mouse model of Fragile X Syndrome.

Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability and is the leading known single-gene cause of autism spectrum disorder. Patients with FXS display varied behavioural deficits that include mild to severe cognitive impairments in addition to mood disorders. Currently, there is no cure for this condition; however, there is an emerging focus on therapies that inhibit mechanistic target of rapamycin (mTOR)-dependent protein synthesis owing to the clinical effectiveness of metformin for alleviating some behavioural symptoms in FXS. Adiponectin (APN) is a neurohormone that is released by adipocytes and provides an alternative means to inhibit mTOR activation in the brain. In these studies, we show that Fmr1 knockout mice, like patients with FXS, show reduced levels of circulating APN and that both long-term potentiation (LTP) and long-term depression (LTD) in the dentate gyrus (DG) are impaired. Brief (20 min) incubation of hippocampal slices in APN (50 nM) was able to rescue both LTP and LTD in the DG and increased both the surface expression and phosphorylation of GluA1 receptors. These results provide evidence for reduced APN levels in FXS playing a role in decreasing bidirectional synaptic plasticity and show that therapies which enhance APN levels may have therapeutic potential for this and related conditions.This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Cage effects on synaptic plasticity and its modulation in a mouse model of fragile X syndrome.

Fragile X syndrome (FXS) is characterized by impairments in executive function including different types of learning and memory. Long-term potentiation (LTP), thought to underlie the formation of memories, has been studied in the Fmr1 mouse model of FXS. However, there have been many discrepancies in the literature with inconsistent use of littermate and non-littermate Fmr1 knockout (KO) and wild-type (WT) control mice. Here, the influence of the breeding strategy (cage effect) on short-term potentiation (STP), LTP, contextual fear conditioning (CFC), expression of N-methyl-d-aspartate receptor (NMDAR) subunits and the modulation of NMDARs, were examined. The largest deficits in STP, LTP and CFC were found in KO mice compared with non-littermate WT. However, the expression of NMDAR subunits was unchanged in this comparison. Rather, NMDAR subunit (GluN1, 2A, 2B) expression was sensitive to the cage effect, with decreased expression in both WT and KO littermates compared with non-littermates. Interestingly, an NMDAR-positive allosteric modulator, UBP714, was only effective in potentiating the induction of LTP in non-littermate KO mice and not the littermate KO mice. These results suggest that commonly studied phenotypes in Fmr1 KOs are sensitive to the cage effect and therefore the breeding strategy may contribute to discrepancies in the literature.This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Incremental induction of NMDAR-STP and NMDAR-LTP in the CA1 area of ventral hippocampal slices relies on graded activation of discrete NMDA receptors.

N-methyl-d-aspartate receptor (NMDAR)-dependent short- and long-term types of potentiation (STP and LTP, respectively) are frequently studied in the CA1 area of dorsal hippocampal slices (DHS). Far less is known about the NMDAR dependence of STP and LTP in ventral hippocampal slices (VHS), where both types of potentiation are smaller in magnitude than in the DHS. Here, we first briefly review our knowledge about the NMDAR dependence of STP and LTP and some other forms of synaptic plasticity. We then show in new experiments that the decay of NMDAR-STP in VHS, similar to dorsal hippocampal NMDAR-STP, is not time- but activity-dependent. We also demonstrate that the induction of submaximal levels of NMDAR-STP and NMDAR-LTP in VHS differs from the induction of saturated levels of plasticity in terms of their sensitivity to subunit-preferring NMDAR antagonists. These data suggest that activation of distinct NMDAR subtypes in a population of neurons results in an incremental increase in the induction of different phases of potentiation with changing sensitivity to pharmacological agents. Differences in pharmacological sensitivity, which arise due to differences in the levels of agonist-evoked biological response, might explain the disparity of the results concerning NMDAR subunit involvement in the induction of NMDAR-dependent plasticity.This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Wavelet transform-based de-noising for two-photon imaging of synaptic Ca2+ transients.

Postsynaptic Ca(2+) transients triggered by neurotransmission at excitatory synapses are a key signaling step for the induction of synaptic plasticity and are typically recorded in tissue slices using two-photon fluorescence imaging with Ca(2+)-sensitive dyes. The signals generated are small with very low peak signal/noise ratios (pSNRs) that make detailed analysis problematic. Here, we implement a wavelet-based de-noising algorithm (PURE-LET) to enhance signal/noise ratio for Ca(2+) fluorescence transients evoked by single synaptic events under physiological conditions. Using simulated Ca(2+) transients with defined noise levels, we analyzed the ability of the PURE-LET algorithm to retrieve the underlying signal. Fitting single Ca(2+) transients with an exponential rise and decay model revealed a distortion of τ(rise) but improved accuracy and reliability of τ(decay) and peak amplitude after PURE-LET de-noising compared to raw signals. The PURE-LET de-noising algorithm also provided a ∼30-dB gain in pSNR compared to ∼16-dB pSNR gain after an optimized binomial filter. The higher pSNR provided by PURE-LET de-noising increased discrimination accuracy between successes and failures of synaptic transmission as measured by the occurrence of synaptic Ca(2+) transients by ∼20% relative to an optimized binomial filter. Furthermore, in comparison to binomial filter, no optimization of PURE-LET de-noising was required for reducing arbitrary bias. In conclusion, the de-noising of fluorescent Ca(2+) transients using PURE-LET enhances detection and characterization of Ca(2+) responses at central excitatory synapses.

Synaptic kainate receptors in CA1 interneurons gate the threshold of theta-frequency-induced long-term potentiation.

Theta oscillations (4-12 Hz) in neuronal networks are known to predispose the synapses involved to plastic changes and may underlie their association with learning behaviors. The lowered threshold for synaptic plasticity during theta oscillations is thought to be due to decreased GABAergic inhibition. Interneuronal kainate receptors (KARs) regulate GABAergic transmission and are implicated in theta activity; however, the physiological significance of this regulation is unknown. In rat hippocampus, we show that during theta activity, there is excitatory postsynaptic drive to CA1 interneurons mediated by KARs. This promotes feedforward inhibition of pyramidal neurons, raising the threshold for induction of theta-burst long-term potentiation. These results identify a novel mechanism whereby the activation of postsynaptic KARs in CA1 interneurons gate changes in synaptic efficacy to a physiologically relevant patterned stimulation.

Plasticity of metabotropic glutamate receptor-dependent long-term depression in the anterior cingulate cortex after amputation.

Long-term depression (LTD) is a key form of synaptic plasticity important in learning and information storage in the brain. It has been studied in various cortical regions, including the anterior cingulate cortex (ACC). ACC is a crucial cortical region involved in such emotion-related physiological and pathological conditions as fear memory and chronic pain. In the present study, we used a multielectrode array system to map cingulate LTD in a spatiotemporal manner within the ACC. We found that low-frequency stimulation (1 Hz, 15 min) applied onto deep layer V induced LTD in layers II/III and layers V/VI. Cingulate LTD requires activation of metabotropic glutamate receptors (mGluRs), while L-type voltage-gated calcium channels and NMDA receptors also contribute to its induction. Peripheral amputation of the distal tail impaired ACC LTD, an effect that persisted for at least 2 weeks. The loss of LTD was rescued by priming ACC slices with activation of mGluR1 receptors by coapplying (RS)-3,5-dihydroxyphenylglycine and MPEP, a form of metaplasticity that involved the activation of protein kinase C. Our results provide in vitro evidence of the spatiotemporal properties of ACC LTD in adult mice. We demonstrate that tail amputation causes LTD impairment within the ACC circuit and that this can be rescued by activation of mGluR1.