Laser microdissection of the dentate gyrus middle molecular layer from a thionin-stained 35 μm section of rat brain.

<p>Intact section (left) and section after microdissection of the middle molecular layer and granule cell layer (right). GCL: granule cell layer; H: hilus; MML: middle molecular layer; CA1: cornu ammonis 1.</p

MicroRNAs, miR-23a-3p and miR-151-3p, Are Regulated in Dentate Gyrus Neuropil following Induction of Long-Term Potentiation <i>In Vivo</i>

<div><p>Translation of synaptic mRNA contributes to alterations in the proteome necessary to consolidate long-term potentiation (LTP), a model of memory processes. Yet, how this process is controlled is not fully resolved. MicroRNAs are non-coding RNAs that negatively regulate gene expression by suppressing translation or promoting mRNA degradation. As specific microRNAs are synaptically located, we hypothesized that they are ideally suited to couple synaptic activation, translational regulation, and LTP persistence. The aim of this study was to identify LTP-regulated microRNAs at or near synapses. Accordingly, LTP was induced unilaterally at perforant path-dentate gyrus synapses in awake adult Sprague-Dawley rats. Five hours later, dentate gyrus middle molecular layer neuropil, containing potentiated synapses, was laser-microdissected. MicroRNA expression profiling, using TaqMan Low Density MicroRNA Microarrays (n = 4), identified eight regulated microRNAs. Subsequent individual TaqMan assays confirmed upregulation of miR-23a-3p (1.30 ± 0.10; p = 0.015) and miR-151-3p (1.17 ± 0.19; p = 0.045) in a second cohort (n = 7). Interestingly, bioinformatic analysis indicated that miR-151-3p and miR-23a-3p regulate synaptic reorganisation and transcription, respectively. In summary, we have demonstrated for the first time that microRNAs are regulated in isolated neuropil following LTP induction <i>in vivo</i>, supporting the hypothesis that synaptic, LTP-responsive microRNAs contribute to LTP persistence via regulation of the synaptic proteome.</p></div

Validation of differential expression of miR-151-3p and miR-23a-3p in the dentate gyrus middle molecular layer 5 h after tetanisation.

<p>Upregulation of miR-151-3p and miR-23a-3p was confirmed by single-plex RT-qPCR (dual criteria: one-tailed Student's t-test p < 0.05; fold change ± 0.15). Expression values: individual and mean fold changes. * p < 0.05; n = 7. Dotted lines indicate cut-off for fold change criterion (± 15%). All data were normalised to miR-301b.</p

LTP regulates microRNA expression in the dentate gyrus middle molecular layer 5 h post-tetanisation.

<p>Regulation of microRNA expression in the middle molecular layer revealed by TaqMan Low Density microRNA Arrays. Eight microRNAs were found to be differentially expressed using dual selection criteria (unadjusted two-tailed Student’s t-test p < 0.05; fold change ± 15%): three were downregulated; five were upregulated. Expression values: individual fold changes and means. Outliers were removed using Grubb’s Test. All data were normalised to miR-301b. Dotted lines indicate cut-off for fold change criterion (± 15%).* p < 0.05; ** p < 0.01; n = 3–4.</p

MiR-151-3p and miR-23a-3p are not differentially expressed in the dentate gyrus granule cell layer 5 h after tetanisation.

<p>Expression values: individual and mean fold changes determined using single-plex RT-qPCR. Dual criteria for differential expression: one-tailed Student's t-test p < 0.05; fold change ± 0.15. N = 7. Dotted lines indicate cut-off for fold change criterion. Data were normalised to U6.</p

Induction of robust LTP at perforant path-granule cell synapses in awake adult rats.

<p>Average (± SEM) field excitatory postsynaptic potential (fEPSP) and population spike (PS) responses expressed as percentage of baseline values. Inset waveforms are averages of 10 sweeps taken just before tetanisation (1), 15–20 min after (2), and 5 h after (3). Calibration bars: 5 ms, 5 mV. 50T: tetanisation paradigm consisting of 50 trains of 400 Hz stimulation.</p

Additional file 1: of Failure of a numerical quality assessment scale to identify potential risk of bias in a systematic review: a comparison study

Table S1. Comparison of different criteria included in the Downs and Black scale and USPSTF system quality assessment tools

Temporal Profiling of Gene Networks Associated with the Late Phase of Long-Term Potentiation <em>In Vivo</em>

<div><p>Long-term potentiation (LTP) is widely accepted as a cellular mechanism underlying memory processes. It is well established that LTP persistence is strongly dependent on activation of constitutive and inducible transcription factors, but there is limited information regarding the downstream gene networks and controlling elements that coalesce to stabilise LTP. To identify these gene networks, we used Affymetrix RAT230.2 microarrays to detect genes regulated 5 h and 24 h (n = 5) after LTP induction at perforant path synapses in the dentate gyrus of awake adult rats. The functional relationships of the differentially expressed genes were examined using DAVID and Ingenuity Pathway Analysis, and compared with our previous data derived 20 min post-LTP induction <em>in vivo</em>. This analysis showed that LTP-related genes are predominantly upregulated at 5 h but that there is pronounced downregulation of gene expression at 24 h after LTP induction. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040538#s2">Analysis</a> of the structure of the networks and canonical pathways predicted a regulation of calcium dynamics via G-protein coupled receptors, dendritogenesis and neurogenesis at the 5 h time-point. By 24 h neurotrophin-NFKB driven pathways of neuronal growth were identified. The temporal shift in gene expression appears to be mediated by regulation of protein synthesis, ubiquitination and time-dependent regulation of specific microRNA and histone deacetylase expression. Together this programme of genomic responses, marked by both homeostatic and growth pathways, is likely to be critical for the consolidation of LTP <em>in vivo.</em></p> </div

LTP-related gene expression 20 min, 5

<p> <b>h and 24 </b><b>h post-LTP induction.</b> Alterations in mRNA transcript number identified 20 min, 5 h and 24 h post-LTP, as detected using Affymetrix RAT 230 2.0 microarrays; note increase in proportion of downregulated genes at 24 h (dual selection criteria: fold change ≥1.15 or ≤0.85; p<0.05).</p

Temporal relationship of LTP-regulated datasets.

<p>(A) Venn Diagram showing 3 datasets largely contain temporally specific genes. (B) Heatmap displaying average gene expression values (expressed as log fold change) of overlapping genes across the timepoints.</p