Metabotropic Glutamate Receptor-dependent Long-term Depression Research Articles

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.

Functional specialization of different PI3K isoforms for the control of neuronal architecture, synaptic plasticity, and cognition.

Neuronal connectivity and activity-dependent synaptic plasticity are fundamental properties that support brain function and cognitive performance. Phosphatidylinositol 3-kinase (PI3K) intracellular signaling controls multiple mechanisms mediating neuronal growth, synaptic structure, and plasticity. However, it is still unclear how these pleiotropic functions are integrated at molecular and cellular levels. To address this issue, we used neuron-specific virally delivered Cre expression to delete either p110α or p110β (the two major catalytic isoforms of type I PI3K) from the hippocampus of adult mice. We found that dendritic and postsynaptic structures are almost exclusively supported by p110α activity, whereas p110β controls neurotransmitter release and metabotropic glutamate receptor-dependent long-term depression at the presynaptic terminal. In addition to these separate functions, p110α and p110β jointly contribute to N-methyl-d-aspartate receptor-dependent postsynaptic long-term potentiation. This molecular and functional specialization is reflected in different proteomes controlled by each isoform and in distinct behavioral alterations for learning/memory and sociability in mice lacking p110α or p110β.

Perinatal exposure to pesticides alters synaptic plasticity signaling and induces behavioral deficits associated with neurodevelopmental disorders

Increasing evidence from animal and epidemiological studies indicates that perinatal exposure to pesticides cause developmental neurotoxicity and may increase the risk for psychiatric disorders such as autism and intellectual disability. However, the underlying pathogenic mechanisms remain largely elusive. This work was aimed at testing the hypothesis that developmental exposure to different classes of pesticides hijacks intracellular neuronal signaling contributing to synaptic and behavioral alterations associated with neurodevelopmental disorders (NDD). Low concentrations of organochlorine (dieldrin, endosulfan, and chlordane) and organophosphate (chlorpyrifos and its oxon metabolite) pesticides were chronically dosed ex vivo (organotypic rat hippocampal slices) or in vivo (perinatal exposure in rats), and then biochemical, electrophysiological, behavioral, and proteomic studies were performed. All the pesticides tested caused prolonged activation of MAPK/ERK pathway in a concentration-dependent manner. Additionally, some of them impaired metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD). In the case of the pesticide chlordane, the effect was attributed to chronic modulation of MAPK/ERK signaling. These synaptic alterations were reproduced following developmental in vivo exposure to chlordane and chlorpyrifos-oxon, and were also associated with prototypical behavioral phenotypes of NDD, including impaired motor development, increased anxiety, and social and memory deficits. Lastly, proteomic analysis revealed that these pesticides differentially regulate the expression of proteins in the hippocampus with pivotal roles in brain development and synaptic signaling, some of which are associated with NDD. Based on these results, we propose a novel mechanism of synaptic dysfunction, involving chronic overactivation of MAPK and impaired mGluR-LTD, shared by different pesticides which may have important implications for NDD.Graphical abstract

PQBP1 promotes translational elongation and regulates hippocampal mGluR-LTD by suppressing eEF2 phosphorylation

Eukaryotic elongation factor 2 (eEF2) mediates translocation of peptidyl-tRNA from the ribosomal A site to the P site to promote translational elongation. Its phosphorylation on Thr56 by its single known kinase eEF2Kinactivates it and inhibits translational elongation. Extensive studies have revealed that different signal cascades modulate eEF2K activity, but whether additional factors regulate phosphorylation of eEF2 remains unclear. Here, we find that the X chromosome-linked intellectual disability protein polyglutamine-bindingprotein 1 (PQBP1) specifically binds to non-phosphorylated eEF2 and suppresses eEF2K-mediated phosphorylation at Thr56. Loss of PQBP1 significantly reduces general protein synthesis by suppressing translational elongation. Moreover, we show that PQBP1 regulates hippocampal metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) and mGluR-LTD-associated behaviors by suppressing eEF2K-mediated phosphorylation. Our results identify PQBP1 as a novel regulator in translational elongation and mGluR-LTD, and this newly revealed regulator in the eEF2K/eEF2 pathway is also an excellent therapeutic target for various disease conditions, such as neural diseases, virus infection, and cancer.

Cystathionine β-Synthase-Derived Hydrogen Sulfide Correlates with Successful Aging in Mice.

Although learning and memory decline are associated with aging, some older individuals show high cognitive function. The mechanism underlying successful aging is not clear, although augmented N-methyl-D-aspartate receptor-independent long-term depression (LTD) has been found in successfully aged rats. We hypothesized that metabotropic glutamate receptor dependent LTD (mGluR-LTD) is associated with successful aging in mice and explored its molecular mechanisms. We divided aged mice into impaired and unimpaired groups and examined mGluR-LTD in the hippocampus. We examined the role of hydrogen sulfide (H2S) in mGluR-LTD establishment in aged mice and investigated the requirement of protein synthesis and intracellular calcium levels. We assessed learning and memory of mice treated with sodium hydrogen sulfide (NaHS) using behavior tests. We found that unimpaired mice elicited larger mGluR-LTD that correlated with H2S production which was blocked by inhibiting cystathionine synthase. Enhanced H2S production of NaHS treatment augmented mGluR-LTD in impaired group. mGluR-LTD establishment required protein synthesis, as well as intracellular calcium, although NaHS treatment leads to reduced sensitivity to calcium chelator 1,2-bis(o-amino phenoxy)ethane-N,N,N',N'-tetraacetic acid treatment. Finally, we found that NaHS treatment enhanced learning and memory of aged mice as indicated by behavioral assessments. Our results indicate that successful aging mice are associated with the function of H2S.

Sevoflurane anesthesia impairs metabotropic glutamate receptor-dependent long-term depression and cognitive functions in senile mice.

Postoperative cognitive dysfunction is often observed in older patients. Previous reports described the link between postoperative cognitive dysfunction and general anesthetics, such as sevoflurane, but the exact mechanism remains unclear. We therefore sought to characterize the effects of sevoflurane on hippocampal-dependent cognitive functions, as well as hippocampal plasticity, and to delineate the underlying mechanisms. Behavioral assays including the novel object recognition test and the Morris water maze test were carried out to assess the cognitive performance of control and sevoflurane-exposed mice. Electrophysiological recordings were carried out to evaluate the sevoflurane-induced changes of synaptic plasticity in the hippocampus. Furthermore, western blot assay was utilized to quantitatively assess the altered protein expression resulting from sevoflurane exposure. Sevoflurane anesthesia impaired cognitive functions, as well as metabotropic glutamate receptor-dependent long-term depression, through elevated surface expression of small conductance calcium-activated potassium type 2 channels. Blockage of calcium-activated potassium type 2 channels reversed the sevoflurane-induced deficits at both cellular and behavioral levels. Sevoflurane anesthesia impaired metabotropic glutamate receptor-dependent long-term depression and thereby affected cognitive functions in old mice. Inhibitory modulators of calcium-activated potassium type 2 channels might prevent cognitive decline elicited by sevoflurane. Geriatr Gerontol Int 2019; 19: 357-362.

Regulation of hippocampal long term depression by Neuroligin 1

Neuroligins (NLGs) are postsynaptic adhesion molecules known to play essential roles in synapse development and maturation, but their effects on synaptic plasticity at mature synapses remain unclear. In this study, we investigate the involvement of NLG1 in hippocampal long-term depression (LTD), a key form of long lasting synaptic plasticity, critical for memory formation and brain disorders, by using mice deficient in the expression of NLG1. We find that although NLG1 homozygous (NLG1−/−) mice show no impairments in either NMDA receptor- (NMDAR-LTD) or metabotropic glutamate receptor-dependent LTD (mGluR-LTD), the heterozygous (NLG1+/−) mice are significantly altered in both forms of LTD characterized by the absence of NMDAR-LTD but enhanced mGluR-LTD. Accordingly, the NLG1+/−, but not the NLG1−/− mice are altered in synaptic proteins, including PSD95, GluA2 and phosphorylated GluA1 at serine 845, all of which are involved in the expression of LTD. The NLG1+/− mice also exhibit autistic-like behaviors including increased grooming and impaired recognition memory. We further show that the expression of NLG3, a close family member of NLG1, is elevated in the NLG1−/−, but not in NLG1+/− mice, suggesting that the lack of LTD deficits in the NLG1−/− mice might be due to the increased NLG3. Our results reveal a gene dosage dependent role for NLG1 in the regulation of LTD and suggest that moderate changes in NLG1 protein level may be sufficient to cause synaptic and behavior deficits in brain disorders where copy number variants and hemizygosity of gene mutations are common.

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.

Acid-sensing ion channel 1a is required for mGlu receptor dependent long-term depression in the hippocampus

Acid-sensing ion channels (ASICs), members of the degenerin/epithelial Na+ channel superfamily, are widely distributed in the mammalian nervous system. ASIC1a is highly permeable to Ca2+ and are thought to be important in a variety of physiological processes, including synaptic plasticity, learning and memory. To further understand the role of ASIC1a in synaptic transmission and plasticity, we investigated metabotropic glutamate (mGlu) receptor-dependent long-term depression (LTD) in the hippocampus. We found that ASIC1a channels mediate a component of LTD in P30-40 animals, since the ASIC1a selective blocker psalmotoxin-1 (PcTx1) reduced the magnitude of LTD induced by application of the group I mGlu receptor agonist (S)-3,5-Dihydroxyphenylglycine (DHPG) or induced by paired-pulse low frequency stimulation (PP-LFS). Conversely, PcTx1 did not affect LTD in P13-18 animals. We also provide evidence that ASIC1a is involved in group I mGlu receptor-induced increase in action potential firing. However, blockade of ASIC1a did not affect DHPG-induced polyphosphoinositide hydrolysis, suggesting the involvement of some other molecular partners in the functional crosstalk between ASIC1a and group I mGlu receptors. Notably, PcTx1 was able to prevent the increase in GluA1 S845 phosphorylation at the post-synaptic membrane induced by group I mGlu receptor activation. These findings suggest a novel function of ASIC1a channels in the regulation of group I mGlu receptor synaptic plasticity and intrinsic excitability.

The Kinase Function of MSK1 Regulates BDNF Signaling to CREB and Basal Synaptic Transmission, But Is Not Required for Hippocampal Long-Term Potentiation or Spatial Memory.

The later stages of long-term potentiation (LTP) in vitro and spatial memory in vivo are believed to depend upon gene transcription. Accordingly, considerable attempts have been made to identify both the mechanisms by which transcription is regulated and indeed the gene products themselves. Previous studies have shown that deletion of one regulator of transcription, the mitogen- and stress-activated kinase 1 (MSK1), causes an impairment of spatial memory. Given the ability of MSK1 to regulate gene expression via the phosphorylation of cAMP response element binding protein (CREB) at serine 133 (S133), MSK1 is a plausible candidate as a prime regulator of transcription underpinning synaptic plasticity and learning and memory. Indeed, prior work has revealed the necessity for MSK1 in homeostatic and experience-dependent synaptic plasticity. However, using a knock-in kinase-dead mouse mutant of MSK1, the current study demonstrates that, while the kinase function of MSK1 is important in regulating the phosphorylation of CREB at S133 and basal synaptic transmission in hippocampal area CA1, it is not required for metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD), two forms of LTP or several forms of spatial learning in the watermaze. These data indicate that other functions of MSK1, such as a structural role for the whole enzyme, may explain previous observations of a role for MSK1 in learning and memory.

PERK regulates Gq protein-coupled intracellular Ca2+ dynamics in primary cortical neurons.

PERK (EIF2AK3) is an ER-resident eIF2α kinase required for behavioral flexibility and metabotropic glutamate receptor-dependent long-term depression via its translational control. Motivated by the recent discoveries that PERK regulates Ca2+ dynamics in insulin-secreting β-cells underlying glucose-stimulated insulin secretion, and modulates Ca2+ signals-dependent working memory, we explored the role of PERK in regulating Gq protein-coupled Ca2+ dynamics in pyramidal neurons. We found that acute PERK inhibition by the use of a highly specific PERK inhibitor reduced the intracellular Ca2+ rise stimulated by the activation of acetylcholine, metabotropic glutamate and bradykinin-2 receptors in primary cortical neurons. More specifically, acute PERK inhibition increased IP3 receptor mediated ER Ca2+ release, but decreased receptor-operated extracellular Ca2+ influx. Impaired Gq protein-coupled intracellular Ca2+ rise was also observed in genetic Perk knockout neurons. Taken together, our findings reveal a novel role of PERK in neurons, which is eIF2α-independent, and suggest that the impaired working memory in forebrain-specific Perk knockout mice may stem from altered Gq protein-coupled intracellular Ca2+ dynamics in cortical pyramidal neurons.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-016-0268-5) contains supplementary material, which is available to authorized users.

PERK Regulates Working Memory and Protein Synthesis-Dependent Memory Flexibility.

PERK (EIF2AK3) is an ER-resident eIF2α kinase required for memory flexibility and metabotropic glutamate receptor-dependent long-term depression, processes known to be dependent on new protein synthesis. Here we investigated PERK’s role in working memory, a cognitive ability that is independent of new protein synthesis, but instead is dependent on cellular Ca2+ dynamics. We found that working memory is impaired in forebrain-specific Perk knockout and pharmacologically PERK-inhibited mice. Moreover, inhibition of PERK in wild-type mice mimics the fear extinction impairment observed in forebrain-specific Perk knockout mice. Our findings reveal a novel role of PERK in cognitive functions and suggest that PERK regulates both Ca2+ -dependent working memory and protein synthesis-dependent memory flexibility.

Actinin-4 Governs Dendritic Spine Dynamics and Promotes Their Remodeling by Metabotropic Glutamate Receptors

Dendritic spines are dynamic, actin-rich protrusions in neurons that undergo remodeling during neuronal development and activity-dependent plasticity within the central nervous system. Although group 1 metabotropic glutamate receptors (mGluRs) are critical for spine remodeling under physiopathological conditions, the molecular components linking receptor activity to structural plasticity remain unknown. Here we identify a Ca(2+)-sensitive actin-binding protein, α-actinin-4, as a novel group 1 mGluR-interacting partner that orchestrates spine dynamics and morphogenesis in primary neurons. Functional silencing of α-actinin-4 abolished spine elongation and turnover stimulated by group 1 mGluRs despite intact surface receptor expression and downstream ERK1/2 signaling. This function of α-actinin-4 in spine dynamics was underscored by gain-of-function phenotypes in untreated neurons. Here α-actinin-4 induced spine head enlargement, a morphological change requiring the C-terminal domain of α-actinin-4 that binds to CaMKII, an interaction we showed to be regulated by group 1 mGluR activation. Our data provide mechanistic insights into spine remodeling by metabotropic signaling and identify α-actinin-4 as a critical effector of structural plasticity within neurons.

Low levels of methyl β-cyclodextrin disrupt GluA1-dependent synaptic potentiation but not synaptic depression.

Methyl-β-cyclodextrin (MβCD) is a reagent that depletes cholesterol and disrupts lipid rafts, a type of cholesterol-enriched cell membrane microdomain. Lipid rafts are essential for neuronal functions such as synaptic transmission and plasticity, which are sensitive to even low doses of MβCD. However, how MβCD changes synaptic function, such as N-methyl-d-aspartate receptor (NMDA-R) activity, remains unclear. We monitored changes in synaptic transmission and plasticity after disrupting lipid rafts with MβCD. At low concentrations (0.5 mg/mL), MβCD decreased basal synaptic transmission and miniature excitatory post-synaptic current without changing NMDA-R-mediated synaptic transmission and the paired-pulse facilitation ratio. Interestingly, low doses of MβCD failed to deplete cholesterol or affect α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA-R) and NMDA-R levels, while clearly reducing GluA1 levels selectively in the synaptosomal fraction. Low doses of MβCD decreased the inhibitory effects of NASPM, an inhibitor for GluA2-lacking AMPA-R. MβCD successfully decreased NMDA-R-mediated long-term potentiation but did not affect the formation of either NMDA-R-mediated or group I metabotropic glutamate receptor-dependent long-term depression. MβCD inhibited de-depression without affecting de-potentiation. These results suggest that MβCD regulates GluA1-dependent synaptic potentiation but not synaptic depression in a cholesterol-independent manner.

Prenatal Ethanol Exposure Alters Synaptic Plasticity in the Dorsolateral Striatum of Rat Offspring via Changing the Reactivity of Dopamine Receptor

Prenatal exposure to high-level ethanol (EtOH) has been reported to produce hyperlocomotion in offspring. Previous studies have demonstrated synaptic plasticity in cortical afferent to the dorsolateral (DL) striatum is involved in the pathogensis of hyperlocomotion. Here, prenatal EtOH-exposed rat offspring were used to investigate whether maternal EtOH exposure affected synaptic plasticity in the DL striatum. We found high-frequency stimulation (HFS) induced a weaker long-term potentiation (LTP) in EtOH rats than that in control rats at postnatal day (PD) 15. The same protocol of HFS induced long-term depression (LTD) in control group but still LTP in EtOH group at PD 30 or PD 40. Furthermore, enhancement of basal synaptic transmission accompanied by the decrease of pair-pulse facilitation (PPF) was observed in PD 30 EtOH offspring. The perfusion with D1-type receptors (D1R) antagonist SCH23390 recovered synaptic transmission and blocked the induction of abnormal LTP in PD 30 EtOH offspring. The perfusion with D2-type receptors (D2R) agonist quinpirole reversed EtOH-induced LTP into D1R- and metabotropic glutamate receptor-dependent LTD. The data provide the functional evidence that prenatal ethanol exposure led to the persistent abnormal synaptic plasticity in the DL striatum via disturbing the balance between D1R and D2R.

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.

A form of synaptically induced metabotropic glutamate receptor-dependent long-term depression that does not require postsynaptic calcium

The calcium control hypothesis posits that postsynaptic calcium increases are required to trigger synaptic plasticity, with large increases inducing LTP and small increases inducing LTD. In CA1 of the hippocampus, however, LTD induced by chemical activation of metabotropic glutamate receptors (agonist-LTD) is independent of increases in postsynaptic calcium. Here we tested whether LTD induced by pairing of presynaptic stimulation with postsynaptic depolarization (synaptic-LTD) is similarly calcium-independent. This protocol induced an NMDA-dependent LTP when paired at 0mV, which was converted to mGluR-dependent LTD when paired at −20mV. The LTD was not blocked by calcium chelation, blockers of L- or T-type voltage-dependent calcium channels, or hyperpolarization to −70mV. We conclude that synaptically induced mGluR-dependent LTD, like agonist induced mGluR LTD, does not require calcium influx for its induction.

Metabotropic glutamate receptor-dependent long-term depression is impaired due to elevated ERK signaling in the ΔRG mouse model of tuberous sclerosis complex

Tuberous sclerosis complex (TSC) and fragile X syndrome (FXS) are caused by mutations in negative regulators of translation. FXS model mice exhibit enhanced metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD). Therefore, we hypothesized that a mouse model of TSC, ΔRG transgenic mice, also would exhibit enhanced mGluR-LTD. We measured the impact of TSC2-GAP mutations on the mTORC1 and ERK signaling pathways and protein synthesis-dependent hippocampal synaptic plasticity in ΔRG transgenic mice. These mice express a dominant/negative TSC2 that binds to TSC1, but has a deletion and substitution mutation in its GAP-domain, resulting in inactivation of the complex. Consistent with previous studies of several other lines of TSC model mice, we observed elevated S6 phosphorylation in the brains of ΔRG mice, suggesting upregulated translation. Surprisingly, mGluR-LTD was not enhanced, but rather was impaired in the ΔRG transgenic mice, indicating that TSC and FXS have divergent synaptic plasticity phenotypes. Similar to patients with TSC, the ΔRG transgenic mice exhibit elevated ERK signaling. Moreover, the mGluR-LTD impairment displayed by the ΔRG transgenic mice was rescued with the MEK–ERK inhibitor U0126. Our results suggest that the mGluR-LTD impairment observed in ΔRG mice involves aberrant TSC1/2-ERK signaling.

Reverse engineering of metabotropic glutamate receptor-dependent long-term depression in the hippocampus

This study focused on metabotropic glutamate receptor-dependent long-term depression (mGluR–LTD) in the hippocampus. This form of LTD is suggested to play a key role in learning, memory and the plasticity of behaviour. Recent advances have started to uncover the underlying mechanisms of mGluR-LTD [1]. However, it is not completely clear how these mechanisms are linked and it is believed that several crucial mechanisms still remain to be revealed. The two main objectives of this study were (i) to quantify the dynamics of mGluR-LTD responses by dynamic data-based models and (ii) to identify underlying dominant processes of mGluR-LTD by applying mathematical system identification methods. In recent years, more and more researchers advocate the use of a top-down modelling approach (reverse engineering) for improving the knowledge of biological systems [2,3]. The drug dihydroxyphenylglycine (DHPG) was used to induce mGluR-LTD in rat brain slices (table ​(table1).1). The drug was applied for different durations (5min, 15min, 2 hours) and in different concentrations (15mM, 30mM). In addition, also different sampling intervals (5min, 30s, 90s) were used. Table 1 Overview of the experiments For the modelling, discrete-time Transfer Functions (TF) models were used. The models described the relation between the DHPG application (input) and the long-term depression responses (output). All models were very accurate (all RT2-values higher than 0,94) and reliably estimated. For a 2 hours application of 30 µM DHPG sampled with a frequency of 1/30s, the time-constant of the mGluR-LTD response was 92s. Thus, the models for high sampling rate indicated that a sampling interval of 30s would be ideal to minimize information loss of the dynamics of mGluR-LTD responses. Interestingly, it was suggested that there are three dominant sub-processes underlying mGluR-LTD: one fast sub-process, one slow sub-process and an immediate sub-process. This study suggests that the dynamic data-based modelling approach can be a valuable tool for reverse engineering of mGluR-dependent LTD responses.

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.