Induction Of mGluR-LTD Research Articles

Stabilization of Spine Synaptopodin by mGluR1 Is Required for mGluR-LTD.

Dendritic spines, actin-rich protrusions forming the postsynaptic sites of excitatory synapses, undergo activity-dependent molecular and structural remodeling. Activation of Group 1 metabotropic glutamate receptors (mGluR1 and mGluR5) by synaptic or pharmacological stimulation, induces LTD, but whether this is accompanied with spine elimination remains unresolved. A subset of telencephalic mushroom spines contains the spine apparatus (SA), an enigmatic organelle composed of stacks of smooth endoplasmic reticulum, whose formation depends on the expression of the actin-bundling protein Synaptopodin. Allocation of Synaptopodin to spines appears governed by cell-intrinsic mechanisms as the relative frequency of spines harboring Synaptopodin is conserved in vivo and in vitro Here we show that expression of Synaptopodin/SA in spines is required for induction of mGluR-LTD at Schaffer collateral-CA1 synapses of male mice. Post-mGluR-LTD, mushroom spines lacking Synaptopodin/SA are selectively lost, whereas spines harboring it are preserved. This process, dependent on activation of mGluR1 but not mGluR5, is conserved in mature mouse neurons and rat neurons of both sexes. Mechanistically, we find that mGluR1 supports physical retention of Synaptopodin within excitatory spine synapses during LTD while triggering lysosome-dependent degradation of the protein residing in dendritic shafts. Together, these results reveal a cellular mechanism, dependent on mGluR1, which enables selective preservation of stronger spines containing Synaptopodin/SA while eliminating weaker ones and potentially countering spurious strengthening by de novo recruitment of Synaptopodin. Overall, our results identify spines with Synaptopodin/SA as the locus of mGluR-LTD and underscore the importance of the molecular microanatomy of spines in synaptic plasticity.SIGNIFICANCE STATEMENT Long-term changes in functional synaptic strength are associated with modification of synaptic connectivity through stabilization or elimination of dendritic spines, the postsynaptic locus of excitatory synapses. How heterogeneous spine microanatomy instructs spine remodeling after long-term synaptic depression (LTD) remains unclear. Metabotropic glutamate receptors mGluR1 and mGluR5 induce a form of LTD critical to circuit function in physiological and disease conditions. Our results identify spines containing the protein Synaptopodin, which enables local assembly of a spine apparatus, as the locus of expression of mGluR-LTD and demonstrate a specific role of mGluR1 in promoting selective loss after mGluR-LTD of mature dendritic spines lacking Synaptopodin/spine apparatus. These findings highlight the fundamental contribution of spine microanatomy in selectively enabling functional and structural plasticity.

Ribosome profiling in mouse hippocampus: plasticity-induced regulation and bidirectional control by TSC2 and FMRP

BackgroundMutations in TSC2 are the most common cause of tuberous sclerosis (TSC), a disorder with a high incidence of autism and intellectual disability. TSC2 regulates mRNA translation required for group 1 metabotropic glutamate receptor-dependent synaptic long-term depression (mGluR-LTD) and behavior, but the identity of mRNAs responsive to mGluR-LTD signaling is largely unknown.MethodsWe utilized Tsc2+/− mice as a mouse model of TSC and prepared hippocampal slices from these animals. We induced mGluR-LTD synaptic plasticity in slices and processed the samples for RNA-seq and ribosome profiling to identify differentially expressed genes in Tsc2+/− and following mGluR-LTD synaptic plasticity.ResultsRibosome profiling reveals that in Tsc2+/− mouse hippocampal slices, the expression of several mRNAs was dysregulated: terminal oligopyrimidine (TOP)-containing mRNAs decreased, while FMRP-binding targets increased. Remarkably, we observed the opposite changes of FMRP binding targets in Fmr1−/y hippocampi. In wild-type hippocampus, induction of mGluR-LTD caused rapid changes in the steady-state levels of hundreds of mRNAs, many of which are FMRP targets. Moreover, mGluR-LTD failed to promote phosphorylation of eukaryotic elongation factor 2 (eEF2) in TSC mice, and chemically mimicking phospho-eEF2 with low cycloheximide enhances mGluR-LTD in TSC mice.ConclusionThese results suggest a molecular basis for bidirectional regulation of synaptic plasticity and behavior by TSC2 and FMRP. Our study also suggests that altered mGluR-regulated translation elongation contributes to impaired synaptic plasticity in Tsc2+/− mice.

G9a/GLP Complex Acts as a Bidirectional Switch to Regulate Metabotropic Glutamate Receptor-Dependent Plasticity in Hippocampal CA1 Pyramidal Neurons.

Metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) is conventionally considered to be solely dependent on local protein synthesis. Given the impact of epigenetics on memory, the intriguing question is whether epigenetic regulation influences mGluR-LTD as well. G9a/GLP histone lysine methyltransferase complex is crucial for brain development and goal-directed learning as well as for drug-addiction. In this study, we analyzed whether the epigenetic regulation by G9a/GLP complex affects mGluR-LTD in CA1 hippocampal pyramidal neurons of 5-7 weeks old male Wistar rats. In hippocampal slices with intact CA1 dendritic regions, inhibition of G9a/GLP activity abolished mGluR-LTD. The inhibition of this complex upregulated the expression of plasticity proteins like PKMζ, which mediated the prevention of mGluR-LTD expression by regulating the NSF-GluA2-mediated trafficking of AMPA receptors towards the postsynaptic site. G9a/GLP inhibition during the induction of mGluR-LTD also downregulated the protein levels of phosphorylated-GluA2 and Arc. Interestingly, G9a/GLP inhibition could not impede the mGluR-LTD when the cell-body was severed. Our study highlights the role of G9a/GLP complex in intact neuronal network as a bidirectional switch; when turned on, it facilitates the expression of mGluR-LTD, and when turned off, it promotes the expression of long-term potentiation.

Coordination between Translation and Degradation Regulates Inducibility of mGluR-LTD.

SUMMARYDendritic protein homeostasis is crucial for most forms of long-term synaptic plasticity, and its dysregulation is linked to a wide range of brain disorders. Current models of metabotropic glutamate receptor mediated long-term depression (mGluR-LTD) suggest that rapid, local synthesis of key proteins is necessary for the induction and expression of LTD. Here, we find that mGluR-LTD can be induced in the absence of translation if the proteasome is concurrently inhibited. We report that enhanced pro-teasomal degradation during the expression of mGluR-LTD depletes dendritic proteins and inhibits subsequent inductions of LTD. Moreover, proteasome inhibition can rescue mGluR-LTD in mice null for the RNA binding protein Sam68, which we show here lack mGluR-dependent translation and LTD. Our study provides mechanistic insights for how changes in dendritic protein abundance regulate mGluR-LTD induction. We propose that Sam68-mediated translation helps to counterbalance degradation, ensuring that protein levels briefly remain above a permissive threshold during LTD induction.

Necessary, but not sufficient: Insights into the mechanisms of mGluR mediated long-term depression from a rat model of early life seizures

Using the rat model of early life seizures (ELS), which has exaggerated mGluR mediated long-term depression of synaptic strength (mGluR-LTD) in adulthood, we probed the signaling cascades underlying mGluR-LTD induction. Several inhibitors completely blocked mGluR-LTD in control but not in ELS rats: the proteasome, the mammalian target of rapamycin (mTOR), S6 kinase (S6K), or L-type voltage-gated calcium channels (L-type VGCC). Inhibition of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) resulted in a near complete block of mGluR-LTD in control rats and a slight reduction of mGluR-LTD in ELS rats. “Autonomous” CaMKII was found to be upregulated in ELS rats, while elevated S6K activity, which is stimulated by mTOR, was described previously. Thus, modulation of each of these factors was necessary for mGluR-LTD induction in control rats, but even their combined, permanent activation in the ELS rats was not sufficient to individually support mGluR-LTD induction following ELS. This implies that while these factors may act sequentially in controls to mediate mGluR-LTD, this is no longer the case after ELS. In contrast, activated ERK was found to be significantly down-regulated in ELS rats. Inhibition of MEK/ERK activation in control rats elevated mGluR-LTD to the exaggerated levels seen in ELS rats. Together, these results elucidate both the mechanisms that persistently enhance mGluR-LTD after ELS and the mechanisms underlying normal mGluR-LTD by providing evidence for multiple, convergent pathways that mediate mGluR-LTD induction. With our prior work, this ties these signaling cascades to the ELS behavioral phenotype that includes abnormal working memory, fear conditioning and socialization.

Long Lasting Protein Synthesis- and Activity-Dependent Spine Shrinkage and Elimination after Synaptic Depression

Neuronal circuits modify their response to synaptic inputs in an experience-dependent fashion. Increases in synaptic weights are accompanied by structural modifications, and activity dependent, long lasting growth of dendritic spines requires new protein synthesis. When multiple spines are potentiated within a dendritic domain, they show dynamic structural plasticity changes, indicating that spines can undergo bidirectional physical modifications. However, it is unclear whether protein synthesis dependent synaptic depression leads to long lasting structural changes. Here, we investigate the structural correlates of protein synthesis dependent long-term depression (LTD) mediated by metabotropic glutamate receptors (mGluRs) through two-photon imaging of dendritic spines on hippocampal pyramidal neurons. We find that induction of mGluR-LTD leads to robust and long lasting spine shrinkage and elimination that lasts for up to 24 hours. These effects depend on signaling through group I mGluRs, require protein synthesis, and activity. These data reveal a mechanism for long lasting remodeling of synaptic inputs, and offer potential insights into mental retardation.

Rapid Synthesis of the X-Linked Mental Retardation Protein OPHN1 Mediates mGluR-Dependent LTD through Interaction with the Endocytic Machinery

Activation of group I metabotropic glutamate receptors leads to long-term depression (mGluR-LTD). Alterations in this form of plasticity have been linked to drug addiction and cognitive disorders. A key characteristic of mGluR-LTD is its dependence on rapid protein synthesis; however, the identities of the proteins mediating LTD remain elusive. Here, we identify the X-linked mental retardation protein OPHN1 as a molecule essential for mGluR-LTD in the hippocampus. mGluR-LTD induction elicits rapid dendritic OPHN1 synthesis, which is dependent on mGluR1 activation and independent of fragile X mental retardation protein (FMRP). This response is essential for mGluR-LTD, as acute blockade of OPHN1 synthesis impedes LTD. mGluR-induced OPHN1 mediates LTD and associated persistent decreases in surface AMPARs via interactions with endophilin A2/3. Importantly, this role of OPHN1 is separable from its effects on basal synaptic strength, which require OPHN1's Rho-GAP activity and interaction with Homer1b/c. Thus, our data establish a role for rapid OPHN1 synthesis in mGluR-LTD.

Insulin Signaling and Autism

ORIGINAL RESEARCH article Front. Endocrinol., 14 October 2011Sec. Cellular Endocrinology https://doi.org/10.3389/fendo.2011.00054

Caveolin-1 knockout mice exhibit impaired induction of mGluR-dependent long-term depression at CA3-CA1 synapses

Group I metabotropic glutamate receptors (mGluR1/5) are important to synaptic circuitry formation during development and to forms of activity-dependent synaptic plasticity. Dysregulation of mGluR1/5 signaling is implicated in some disorders of neurodevelopment, including fragile X syndrome, the most common inherited form of intellectual disabilities and leading cause of autism. Site(s) in the intracellular loops of mGluR1/5 directly bind caveolin-1, an adaptor protein that associates with membrane rafts. Caveolin-1 is the main coat component of caveolae and organizes macromolecular signaling complexes with effector proteins and membrane receptors. We report that long-term depression (LTD) elicited by a single application of the group I mGluR selective agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) was markedly attenuated at Schaffer collateral-CA1 synapses of mice lacking caveolin-1 (Cav1(-/-)), as assessed by field recording. In contrast, multiple applications of DHPG produced LTD comparable to that in WT mice. Passive membrane properties, basal glutamatergic transmission and NMDA receptor (NMDAR)-dependent LTD were unaltered. The remaining LTD was reduced by anisomycin, an inhibitor of protein synthesis, by U0126, an inhibitor of MEK1/2 kinases, and by rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), suggesting mediation by the same mechanisms as in WT. mGluR1/5-dependent activation (phosphorylation) of MEK and extracellular signal-regulated kinase (ERK1/2) was altered in Cav1(-/-) mice; basal phosphorylation was increased, but a single application of DHPG had no further effect, and after DHPG, phosphorylation was similar in WT and Cav1(-/-) mice. Taken together, our findings suggest that caveolin-1 is required for normal coupling of mGluR1/5 to downstream signaling cascades and induction of mGluR-LTD.

Regulation of microRNA Expression by Induction of Bidirectional Synaptic Plasticity

Activity-induced protein synthesis is critical for long-lasting synaptic plasticity and subject to tight controls. MicroRNAs (miRNAs) are negative regulators of mRNA translation, but their role during synaptic plasticity is not clear. In this study, we have investigated how induction of long-term potentiation (LTP) and long-term depression (LTD) regulates the expression of miRNAs. Using miRNA arrays, we determined the temporal expression profiles of 62 hippocampal miRNAs following induction of chemical LTP (C-LTP) and metabotropic glutamate receptor-dependent LTD (mGluR-LTD). Several striking features were observed. First, C-LTP or mGluR-LTD induction changed the expression levels of most hippocampal miRNAs. Second, the majority of miRNAs regulated by C-LTP or mGluR-LTD induction followed a similar temporal expression profile. Third, most miRNAs were regulated by both C-LTP and mGluR-LTD induction, but displayed distinct expression dynamics. Fourth, many miRNAs were upregulated at specific time points C-LTP and mGluR-LTD induction, suggesting that C-LTP and mGluR-LTD induction elicits miRNA-mediated suppression of mRNA translation. We propose that the upregulated miRNA expression provides a mechanism to prevent excess protein synthesis during the expression of synaptic plasticity. The extensive regulation of miRNA expression by C-LTP and mGluR-LTD induction suggests a critical role of miRNAs in synaptic plasticity.

Rapid Translation of Arc/Arg3.1 Selectively Mediates mGluR-Dependent LTD through Persistent Increases in AMPAR Endocytosis Rate

Salient stimuli that modify behavior induce transcription of activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) and transport Arc mRNA into dendrites, suggesting that local Arc translation mediates synaptic plasticity that encodes such stimuli. Here, we demonstrate that long-term synaptic depression (LTD) in hippocampal neurons induced by group 1 metabotropic glutamate receptors (mGluRs) relies on rapid translation of Arc. mGluR-LTD induction causes long-term increases in AMPA receptor endocytosis rate and dendritic synthesis of Arc, a component of the AMPAR endocytosis machinery. Knockdown of Arc prevents mGluRs from triggering AMPAR endocytosis or LTD, and acute blockade of new Arc synthesis with antisense oligonucleotides blocks mGluR-LTD and AMPAR trafficking. In contrast, LTD induced by NMDA receptors does not persistently alter AMPAR endocytosis rate, induce Arc synthesis, or require Arc protein. These data demonstrate a role for local Arc synthesis specifically in mGluR-LTD and suggest that mGluR-LTD may be one consequence of Arc mRNA induction during experience.

Rapid Synthesis and Synaptic Insertion of GluR2 for mGluR-LTD in the Ventral Tegmental Area

The activation of metabotropic glutamate receptors (mGluRs) leads to long-term depression (mGluR-LTD) at many synapses of the brain. The induction of mGluR-LTD is well characterized, whereas the mechanisms underlying its expression remain largely elusive. mGluR-LTD in the ventral tegmental area (VTA) efficiently reverses cocaine-induced strengthening of excitatory inputs onto dopamine neurons. We show that mGluR-LTD is expressed by an exchange of GluR2-lacking AMPA receptors for GluR2-containing receptors with a lower single-channel conductance. The synaptic insertion of GluR2 depends on de novo protein synthesis via rapid messenger RNA translation of GluR2. Regulated synthesis of GluR2 in the VTA is therefore required to reverse cocaine-induced synaptic plasticity.

Developmental dependence, the role of the kinases p38 MAPK and PKC, and the involvement of tumor necrosis factor-R1 in the induction of mGlu-5 LTD in the dentate gyrus

The mechanisms of mGluR-LTD were studied in the dentate gyrus in vitro. The most effective protocol for inducing mGluR-LTD in 6–8 week animals was brief high frequency stimulation (HFS) applied in the presence of the NMDAR antagonist AP5. Evidence for HFS inducing LTD via activation of perisynaptically located mGluRs was established, as an inhibitor of glutamate transporter potentiated HFS-LTD. HFS-LTD was mainly mediated by activation of mGluR5, although a partial involvement of mGluR1 was found. (RS)-3,5-Dihydroxyphenylglycine (DHPG) also induced LTD, but in an age dependent manner, being large in 2 week animals but absent in 6–8 week animals. DHPG-LTD in the dentate gyrus also had a much slower rise time than that in CA1, and unlike CA1, the expression/maintenance of mGluR-LTD was not inhibited by mGluR antagonists. The use of pharmacological inhibitors showed that the induction of HFS-LTD was partially dependent upon activation of L-type Ca channels, release of Ca from ryanodine receptor-sensitive intracellular Ca stores, and the kinases p38 mitogen-activated protein kinase (MAPK), protein kinase C (PKC), but not c-Jun N-terminal kinase or COX-2. Evidence for the involvement of tumor necrosis factor-receptor 1 (TNF-R1) in the induction of mGluR-LTD was presented in the present study, with both HFS-mGluR-LTD and DHPG-LTD being absent in mutant mice null for TNF-R1.

12-Lipoxygenase Metabolites of Arachidonic Acid Mediate Metabotropic Glutamate Receptor-Dependent Long-Term Depression at Hippocampal CA3-CA1 Synapses

Arachidonic acid metabolites have been proposed as signaling molecules in hippocampal long-term potentiation (LTP) and long-term depression (LTD) for >15 years. However, the functional role of these molecules remains controversial. Here we used a multidisciplinary biochemical, electrophysiological, and genetic approach to examine the function of the 12-lipoxygenase metabolites of arachidonic acid in long-term synaptic plasticity at CA3-CA1 synapses. We found that the 12-lipoxygenase pathway is required for the induction of metabotropic glutamate receptor-dependent LTD (mGluR-LTD), but is not required for LTP: (1) Hippocampal homogenates were capable of synthesizing the 12-lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxyeicosa-5Z,8Z,10E,14Z-tetraenoic acid (HETE). (2) Stimulation protocols that induce mGluR-LTD lead to a release of 12-(S)-HETE from acute hippocampal slices. (3) A mouse in which the leukocyte-type 12-lipoxygenase (the neuronal isoform) was deleted through homologous recombination was deficient in mGluR-LTD, but showed normal LTP. (4) Pharmacological inhibition of 12-lipoxygenase also blocked induction of mGluR-LTD. (5) Finally, direct application of 12(S)-HPETE, but not 15(S)-HPETE, to hippocampal slices induced a long-term depression of synaptic transmission that mimicked and occluded mGluR-LTD induced by synaptic stimulation. Thus, 12(S)-hydroperoxyeicosa-5Z, 8Z, 10E, 14Z-tetraenoic acid (12(S)-HPETE), a 12-lipoxygenase metabolite of arachidonic acid, satisfies all of the criteria of a messenger molecule that is actively recruited for the induction of mGluR-LTD.

Synaptically stimulated induction of group i metabotropic glutamate receptor-dependent long-term depression and depotentiation is inhibited by prior activation of metabotropic glutamate receptors and PROTEIN KINASE C

We have investigated metaplasticity of the group I metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD) and depotentiation (DP) induced by physiological synaptic stimulation in the medial perforant path of the dentate gyrus in vitro. Group I mGluR-LTD/DP was inhibited by prior preconditioning brief high frequency stimulation (HFS) if the preconditioning HFS induced long-term potentiation (LTP) or if the induction of LTP was inhibited by an NMDA receptor antagonist. The inhibitory effect of the preconditioning HFS on LTD/DP was dependent upon activation of mGluRs, as it was blocked by the presence of the mGluR antagonist ( S)-α-methyl-4-carboxyphenylglycine during the preconditioning stimulation. The inhibitory effect of the preconditioning HFS involved stimulation of PKC, as the presence of the PKC inhibitor bisindolylmaleimide (BIS) during the preconditioning stimulation prevented the inhibitory effect of such preconditioning stimulation. Activation of PKC was also necessary for the induction of mGluR-LTD itself, as the PKC inhibitor BIS prevented the induction of the mGluR-LTD. We suggest that the physiological stimulation of mGluRs by the preconditioning stimulation produces a PKC-dependent inactivation of subsequent group I mGluR functioning and thereby an inhibition of induction of group I mGluR-dependent LTD/DP induction.

Altered Presynaptic Vesicle Release and Cycling during mGluR-Dependent LTD

The site of modification of synaptic transmission during long-term plasticity in the mammalian hippocampus remains controversial. Here we used a fluorescent marker of presynaptic activity, FM 1-43, to directly image presynaptic function during metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) at CA3-CA1 excitatory synapses in acute hippocampal slices. We found a significant decrease in the rate of FM 1-43 release in response to synaptic stimulation following induction of mGluR-LTD, providing direct evidence for altered presynaptic function. Moreover, we found that mGluR-LTD causes several changes in FM dye release properties that are consistent with a change in the mode of vesicle cycling, possibly involving a switch from a full fusion mode of release to a “kiss-and-run” mode of release through the transient opening of a fusion pore.