Reciprocal regulation of microRNA and mRNA profiles in neuronal development and synapse formation.

BACKGROUND: Synapse formation and the development of neural networks are known to be controlled by a coordinated program of mRNA synthesis. microRNAs are now recognized to be important regulators of mRNA translation and stability in a wide variety of organisms. While specific microRNAs are known to be involved in neural development, the extent to which global microRNA and mRNA profiles are coordinately regulated in neural development is unknown. RESULTS: We examined mouse primary neuronal cultures, analyzing microRNA and mRNA expression. Three main developmental patterns of microRNA expression were observed: steady-state levels, up-regulated and down-regulated. Co-expressed microRNAs were found to have related target recognition sites and to be encoded in distinct genomic locations. A number of 43 differentially expressed miRNAs were located in five genomic clusters. Their predicted mRNA targets show reciprocal levels of expression. We identified a set of reciprocally expressed microRNAs that target mRNAs encoding postsynaptic density proteins and high-level steady-state microRNAs that target non-neuronal low-level expressed mRNAs. CONCLUSION: We characterized hundreds of miRNAs in neuronal culture development and identified three major modes of miRNA expression. We predict these miRNAs to regulate reciprocally expressed protein coding genes, including many genes involved in synaptogenesis. The identification of miRNAs that target mRNAs during synaptogenesis indicates a new level of regulation of the synapse

Recording long-term potentiation of synaptic transmission by three-dimensional multi-electrode arrays

BACKGROUND: Multi-electrode arrays (MEAs) have become popular tools for recording spontaneous and evoked electrical activity of excitable tissues. The majority of previous studies of synaptic transmission in brain slices employed MEAs with planar electrodes that had limited ability to detect signals coming from deeper, healthier layers of the slice. To overcome this limitation, we used three-dimensional (3D) MEAs with tip-shaped electrodes to probe plasticity of field excitatory synaptic potentials (fEPSPs) in the CA1 area of hippocampal slices of 129S5/SvEvBrd and C57BL/6J-Tyr(C-Brd )mice. RESULTS: Using 3D MEAs, we were able to record larger fEPSPs compared to signals measured by planar MEAs. Several stimulation protocols were used to induce long-term potentiation (LTP) of synaptic responses in the CA1 area recorded following excitation of Schäffer collateral/commissural fibres. Either two trains of high frequency tetanic stimulation or three trains of theta-burst stimulation caused a persistent, pathway specific enhancement of fEPSPs that remained significantly elevated for at least 60 min. A third LTP induction protocol that comprised 150 pulses delivered at 5 Hz, evoked moderate LTP if excitation strength was increased to 1.5× of the baseline stimulus. In all cases, we observed a clear spatial plasticity gradient with maximum LTP levels detected in proximal apical dendrites of pyramidal neurones. No significant differences in the manifestation of LTP were observed between 129S5/SvEvBrd and C57BL/6J-Tyr(C-Brd )mice with the three protocols used. All forms of plasticity were sensitive to inhibition of N-methyl-D-aspartate (NMDA) receptors. CONCLUSION: Principal features of LTP (magnitude, pathway specificity, NMDA receptor dependence) recorded in the hippocampal slices using MEAs were very similar to those seen in conventional glass electrode experiments. Advantages of using MEAs are the ability to record from different regions of the slice and the ease of conducting several experiments on a multiplexed platform which could be useful for efficient screening of novel transgenic mice

A Neuronal Transcriptome Response Involving Stress Pathways is Buffered by Neuronal microRNAs.

A single microRNA (miRNA) can inhibit a large number of mRNA transcripts. This widespread regulatory function has been experimentally demonstrated for a number of miRNAs. However, even when a multitude of targets is confirmed, function of a miRNA is frequently interpreted through a prism of a handful arbitrarily selected "interesting" targets. In this work we first show that hundreds of transcripts with target sites for two miRNAs expressed endogenously in neurons, miR-124 and miR-434-3p, are coordinately upregulated in a variety of neuronal stresses. This creates a landscape where these two miRNAs can exert their widespread inhibitory potential on stress-induced transcripts. Next, we experimentally demonstrate that overexpression of these two miRNAs indeed significantly inhibits expression of hundreds of stress-induced transcripts, thus confirming that these transcripts are enriched in true targets of examined miRNAs. A number of miRNAs were previously shown to have important roles in the regulation of stress responses, and our results suggest that these roles should be understood in light of a wide spread activation of miRNA targets during stresses. Importantly, a popular cationic lipid transfection reagent triggers such induction of miRNA targets. Therefore, when a transfection paradigm is employed to study miRNA function, the results of such studies should be interpreted with consideration for the inadvertent induction of miRNA targets.This work was supported by the Darwin Trust of Edinburgh, Wellcome Trust and EMBL

Quantitative differences in developmental profiles of spontaneous activity in cortical and hippocampal cultures.

BACKGROUND: Neural circuits can spontaneously generate complex spatiotemporal firing patterns during development. This spontaneous activity is thought to help guide development of the nervous system. In this study, we had two aims. First, to characterise the changes in spontaneous activity in cultures of developing networks of either hippocampal or cortical neurons dissociated from mouse. Second, to assess whether there are any functional differences in the patterns of activity in hippocampal and cortical networks. RESULTS: We used multielectrode arrays to record the development of spontaneous activity in cultured networks of either hippocampal or cortical neurons every 2 or 3 days for the first month after plating. Within a few days of culturing, networks exhibited spontaneous activity. This activity strengthened and then stabilised typically around 21 days in vitro. We quantified the activity patterns in hippocampal and cortical networks using 11 features. Three out of 11 features showed striking differences in activity between hippocampal and cortical networks: (1) interburst intervals are less variable in spike trains from hippocampal cultures; (2) hippocampal networks have higher correlations and (3) hippocampal networks generate more robust theta-bursting patterns. Machine-learning techniques confirmed that these differences in patterning are sufficient to classify recordings reliably at any given age as either hippocampal or cortical networks. CONCLUSIONS: Although cultured networks of hippocampal and cortical networks both generate spontaneous activity that changes over time, at any given time we can reliably detect differences in the activity patterns. We anticipate that this quantitative framework could have applications in many areas, including neurotoxicity testing and for characterising the phenotype of different mutant mice. All code and data relating to this report are freely available for others to use.PC and AM were supported by the Wellcome Trust Genes to Cognition programme. PC received additional support from the Biotechnology and Biological Sciences Research Council (BB/H008608/1). EC was supported by a Wellcome Trust PhD studentship and Cambridge Biomedical Research Centre studentship. SJE was supported by an Engineering and Physical Sciences Research Council grant (EP/E002331/1).This is the final published version. It first appeared at http://link.springer.com/article/10.1186%2Fs13064-014-0028-0

Evolution of NMDA receptor cytoplasmic interaction domains: implications for organisation of synaptic signalling complexes

<p>Abstract</p> <p>Background</p> <p>Glutamate gated postsynaptic receptors in the central nervous system (CNS) are essential for environmentally stimulated behaviours including learning and memory in both invertebrates and vertebrates. Though their genetics, biochemistry, physiology, and role in behaviour have been intensely studied <it>in vitro </it>and <it>in vivo</it>, their molecular evolution and structural aspects remain poorly understood. To understand how these receptors have evolved different physiological requirements we have investigated the molecular evolution of glutamate gated receptors and ion channels, in particular the <it>N</it>-methyl-<it>D</it>-aspartate (NMDA) receptor, which is essential for higher cognitive function. Studies of rodent NMDA receptors show that the C-terminal intracellular domain forms a signalling complex with enzymes and scaffold proteins, which is important for neuronal and behavioural plasticity</p> <p>Results</p> <p>The vertebrate NMDA receptor was found to have subunits with C-terminal domains up to 500 amino acids longer than invertebrates. This extension was specific to the NR2 subunit and occurred before the duplication and subsequent divergence of NR2 in the vertebrate lineage. The shorter invertebrate C-terminus lacked vertebrate protein interaction motifs involved with forming a signaling complex although the terminal PDZ interaction domain was conserved. The vertebrate NR2 C-terminal domain was predicted to be intrinsically disordered but with a conserved secondary structure.</p> <p>Conclusion</p> <p>We highlight an evolutionary adaptation specific to vertebrate NMDA receptor NR2 subunits. Using <it>in silico </it>methods we find that evolution has shaped the NMDA receptor C-terminus into an unstructured but modular intracellular domain that parallels the expansion in complexity of an NMDA receptor signalling complex in the vertebrate lineage. We propose the NR2 C-terminus has evolved to be a natively unstructured yet flexible hub organising postsynaptic signalling. The evolution of the NR2 C-terminus and its associated signalling complex may contribute to species differences in behaviour and in particular cognitive function.</p

Proteomic analysis of NMDA receptor–adhesion protein signaling complexes

N-methyl-D-aspartate receptors (NMDAR) mediate long-lasting changes in synapse strength via downstream signaling pathways. We report proteomic characterization with mass spectrometry and immunoblotting of NMDAR multiprotein complexes (NRC) isolated from mouse brain. The NRC comprised 77 proteins organized into receptor, adaptor, signaling, cytoskeletal and novel proteins, of which 30 are implicated from binding studies and another 19 participate in NMDAR signaling. NMDAR and metabotropic glutamate receptor subtypes were linked to cadherins and L1 cell-adhesion molecules in complexes lacking AMPA receptors. These neurotransmitter– adhesion receptor complexes were bound to kinases, phosphatases, GTPase-activating proteins and Ras with effectors including MAPK pathway components. Several proteins were encoded by activity-dependent genes. Genetic or pharmacological interference with 15 NRC proteins impairs learning and with 22 proteins alters synaptic plasticity in rodents. Mutations in three human genes (NF1, Rsk-2, L1) are associated with learning impairments, indicating the NRC also participates in human cognition

PSD95 nanoclusters are postsynaptic building blocks in hippocampus circuits

The molecular features of synapses in the hippocampus underpin current models of learning and cognition. Although synapse ultra-structural diversity has been described in the canonical hippocampal circuitry, our knowledge of sub-synaptic organisation of synaptic molecules remains largely unknown. To address this, mice were engineered to express Post Synaptic Density 95 protein (PSD95) fused to either eGFP or mEos2 and imaged with two orthogonal super-resolution methods: gated stimulated emission depletion (g-STED) microscopy and photoactivated localisation microscopy (PALM). Large-scale analysis of ~100,000 synapses in 7 hippocampal sub-regions revealed they comprised discrete PSD95 nanoclusters that were spatially organised into single and multi-nanocluster PSDs. Synapses in different sub-regions, cell-types and locations along the dendritic tree of CA1 pyramidal neurons, showed diversity characterised by the number of nanoclusters per synapse. Multi-nanocluster synapses were frequently found in the CA3 and dentate gyrus sub-regions, corresponding to large thorny excrescence synapses. Although the structure of individual nanoclusters remained relatively conserved across all sub-regions, PSD95 packing into nanoclusters also varied between sub-regions determined from nanocluster fluorescence intensity. These data identify PSD95 nanoclusters as a basic structural unit, or building block, of excitatory synapses and their number characterizes synapse size and structural diversity.Support from the Wellcome Trust, Medical Research Council, European Commission.This is the final version of the article. It first appeared from Nature Publishing Group via https://doi.org/10.1038/srep2462

PSD95 nanoclusters are postsynaptic building blocks in hippocampus circuits.

The molecular features of synapses in the hippocampus underpin current models of learning and cognition. Although synapse ultra-structural diversity has been described in the canonical hippocampal circuitry, our knowledge of sub-synaptic organisation of synaptic molecules remains largely unknown. To address this, mice were engineered to express Post Synaptic Density 95 protein (PSD95) fused to either eGFP or mEos2 and imaged with two orthogonal super-resolution methods: gated stimulated emission depletion (g-STED) microscopy and photoactivated localisation microscopy (PALM). Large-scale analysis of ~100,000 synapses in 7 hippocampal sub-regions revealed they comprised discrete PSD95 nanoclusters that were spatially organised into single and multi-nanocluster PSDs. Synapses in different sub-regions, cell-types and locations along the dendritic tree of CA1 pyramidal neurons, showed diversity characterised by the number of nanoclusters per synapse. Multi-nanocluster synapses were frequently found in the CA3 and dentate gyrus sub-regions, corresponding to large thorny excrescence synapses. Although the structure of individual nanoclusters remained relatively conserved across all sub-regions, PSD95 packing into nanoclusters also varied between sub-regions determined from nanocluster fluorescence intensity. These data identify PSD95 nanoclusters as a basic structural unit, or building block, of excitatory synapses and their number characterizes synapse size and structural diversity.Support from the Wellcome Trust, Medical Research Council, European Commission.This is the final version of the article. It first appeared from Nature Publishing Group via https://doi.org/10.1038/srep2462

Supramolecular organization of NMDA receptors and the postsynaptic density

The postsynaptic density (PSD) of all vertebrate species share a highly complex proteome with ∼1000 conserved proteins that function as sophisticated molecular computational devices. Here, we review recent studies showing that this complexity can be understood in terms of the supramolecular organization of proteins, which self-assemble within a hierarchy of different length scales, including complexes, supercomplexes and nanodomains. We highlight how genetic and biochemical approaches in mice are being used to uncover the native molecular architecture of the synapse, revealing hitherto unknown molecular structures, including highly selective mechanisms for specifying the assembly of NMDAR-MAGUK supercomplexes. We propose there exists a logical framework that precisely dictates the subunit composition of synaptic complexes, supercomplexes, and nanodomains in vivo