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

Evolution of GluN2A/B cytoplasmic domains diversified vertebrate synaptic plasticity and behavior

Two genome duplications early in the vertebrate lineage expanded gene families, including GluN2 subunits of the NMDA receptor. Diversification between the four mammalian GluN2 proteins occurred primarily at their intracellular C−terminal domains (CTDs). To identify shared ancestral functions and diversified subunit−specific functions, we exchanged the exons encoding the GluN2A (also known as Grin2a) and GluN2B (also known as Grin2b) CTDs in two knock−in mice and analyzed the mice's biochemistry, synaptic physiology, and multiple learned and innate behaviors. The eight behaviors were genetically separated into four groups, including one group comprising three types of learning linked to conserved GluN2A/B regions. In contrast, the remaining five behaviors exhibited subunit−specific regulation. GluN2A/B CTD diversification conferred differential binding to cytoplasmic MAGUK proteins and differential forms of long−term potentiation. These data indicate that vertebrate behavior and synaptic signaling acquired increased complexity from the duplication and diversification of ancestral GluN2 gene

Mouse models of 17q21.31 rearrangements.

<p><b>(A)</b> Top: haplotype H1 of the human 17q21.31 region. All genomic positions are given according to the UCSC human genome browser GRCh38/hg38. Bottom: 17q21.31 syntenic region on mouse chromosome 11E1. All genomic positions are given according to UCSC mouse genome browser GRCm38/mm10. <b>(B)</b> Strategy for <i>in vivo</i> Cre-mediated recombination and targeted meiotic recombination (TAMERE) crossing strategy. LoxP sites were inserted upstream of <i>Arf2</i> and downstream of <i>Kansl1</i>. The breeding strategy aimed to have trans-loxer females expressing the <i>Hprt</i>^{<i>tm1(cre)Mnn</i>} transgene and carrying the two loxP sites in a <i>trans</i> configuration. The last step consisted of mating trans-loxer females with wt males to generate progeny carrying the deletion (<i>Del/+</i>) or the duplication (<i>Dup/+</i>) of the <i>Arf2-Kansl1</i> region. <i>Del/+</i> and <i>Dup/+</i> animals were crossed together to generate <i>Del-Dup</i> cohorts. <b>(C)</b> Molecular validation. PCR products specific for the <i>Del</i> and <i>Dup</i> alleles are 448-bp and 653-bp respectively. <b>(D)</b> Evolution of body weight (g) of adult animals (F_(3,300) = 5.529, <i>P</i> = 0.002; <i>Del/+</i> vs wt: P = 0.101, <i>Dup/+</i> vs wt: <i>P</i> = 0.002, <i>Del/Dup</i> vs wt: P = 0.109). <b>(E)</b> Body length (distance from the snout to the tail base) of 20-week old animals (H_{(3, 55)} = 28.036, <i>P</i> < 0.001; <i>Del/+</i> vs wt: <i>P</i> = 0.004, <i>Dup/+</i> vs wt: <i>P</i> < 0.001, <i>Del/Dup</i> vs wt: <i>P</i> = 0.003). <b>(F)</b> Body fat percentage of 20-week-old animals measured by qNMR (H_{(3, 55)} = 8.120, <i>P</i> = 0.044; <i>Del/+</i> vs wt: <i>P</i> = 0.027). Data are represented as the mean ± s.e.m. Cohort used included 18 <i>Del/+</i>, 24 wt, 11 <i>Del/Dup</i>, and 11 <i>Dup/+</i> animals. <b>(A)</b> Repeated Measures ANOVA “genotype” analysis, Tukey's <i>post hoc</i> test. <b>(B, C, E, F)</b> Kruskal-Wallis analysis, Mann-Whitney <i>U</i> test. *<i>P</i> < 0.05 vs wt, **<i>P</i> < 0.01 vs wt, ***<i>P</i> < 0.001 vs wt, ^# <i>P</i> < 0.05 vs all other groups.</p

Epigenetic profiling in the hippocampus of KdV mouse models demonstrated non-neuronal and neuronal cell-specific alterations.

<p>(<b>A</b>) H3K4me3 pattern in the deleted region and its flanking sequences. Graph with a schematic representation of H3k4me3 peaks (averaged over three biological replicates) at the promoters of the genes in the 17q21.31 deleted region and flanking regions. Peaks within the deletion show half intensity as compared to wt, while peaks in the surrounding regions show the same intensity and patterning, thus confirming the absence of nonspecific-effects. (<b>B</b>) Overview of promoter deregulations. For each mouse model (<i>Kansl1</i>^<i>+/-</i> or <i>Del/+</i>) 3 wt biological replicas and 3 mutant biological replicas were generated for H3K4me3 ChIP-seq. DEseq2 algorithm was used to call the significant deregulations (<i>P</i> value<0.01) in the promoters. False positives, as detected by comparing the two groups of WT samples, can be estimated to only 4%. Given that no consistent changes were found among WT sets we opted to collapse all the 6 wts in one group and use it as the common control for both <i>Kansl1</i>^<i>+/-</i> and <i>Del/+</i> samples, thus increasing the sensitivity and detecting more dysregulation (right histograms) (<b>C</b>) Clustering of Pearson correlation calculated over the total dysregulated promoters clearly segregates the different conditions. (<b>D</b>) Cell types of <i>Del/+</i> up-regulated genes. The Stacked histogram represents the enrichment of cell-type specific markers in the <i>Del/+</i> UP-regulated genes. The strongest enrichment is for CA1 pyramidal neurons markers, followed by genes specific to somatosensory cortex pyramidal neurons, astrocytes and interneurons. A milder, but still significant, enrichment was found for endothelial cells. Together, these results suggest that <i>Del/+</i> deletion causes perturbations that are cell-type specific. (<b>E</b>) Overview of <i>Del/+</i> misregulation in the <i>Kansl1</i>^<i>+/-</i> model. Pie charts summarize the behavior of <i>Del/+</i> deregulated genes in the <i>Kansl1</i>^<i>+/-</i> model to evaluate the extent to which <i>Kansl1</i> alone is able to mimic the <i>Del/+</i> misregulations. To better describe the shades of gray always inherent to the <i>P</i> values, we classified the strength of <i>Kansl1</i>^<i>+/-</i> deregulation in four classes, ranging from no-change to the same intensity of <i>Del/+</i>. We could not detect any ‘reversal’ in the deregulations (i.e. genes up-regulated in one mutant being down-regulated in the other and vice versa) (<b>E</b>) Clustering of the <i>Del/</i>+ and <i>Kansl1</i>^<i>+/-</i> deregulated genes. The heat map includes all the genes that are significantly UP-regulated in either of the models. Enrichment of neuronal markers are based on published work. (<b>F</b>) Study of cell-type markers. The heat map represents the significance of the enrichments for specific markers in the 4 different clusters of section E. (<b>G</b>) Study of Cluster GO enrichments. The heat map represents the significance of the GO enrichments in the 4 clusters of section E. (<b>I</b>) overview of social behavior genes.</p

Behavioral characterization of <i>Del-Dup</i> cohorts.

<p><b>(A)</b> Circadian activity test. Graphs plot the spontaneous ambulatory activity (count) and the vertical activity/rears (count) during dark and light phases. Del/+ mice showed reduced ambulatory activity during the dark phase (F_(3,46) = 5.791, <i>P</i> = 0.002; <i>Del/+</i> vs wt: <i>P</i> = 0.002) and the light phase (F_(3,46) = 4.260, <i>P</i> = 0.010; <i>Del/+</i> vs wt: <i>P</i> = 0.006) as well as reduced rearing behavior during the light phase (H_{(3, 46)} = 12.861, <i>P</i> = 0.005; <i>Del/+</i> vs wt: <i>P</i> = 0.002) <b>(B)</b> Open field test. Distance travelled (m), and vertical activity/rears (count) over 30 min of testing. <b>(C)</b> Novel object recognition test. Discrimination index was calculated as the ratio of time spent exploring the novel object vs the total time for object sniffing in the choice trial after a 3 h retention delay. The dashed line denotes a chance level of 50% (F_(3,43) = 3.081, <i>P</i> = 0.037; <i>Del/+</i> vs wt: <i>P</i> = 0.040). <b>(D)</b> Fear conditioning test. Plots represent the percentage of time spent freezing during test sessions. The 6-min context session was run 24 h after conditioning. The 8-min cue session was performed 5 h after the context session. A sequence of 2-min without cue and 2 min with light/auditory conditioning stimulus was repeated twice. <b>(E)</b> Cued fear extinction. Independent group of <i>Del /+</i> and <i>Dup/+</i> mutant mice with their respective wt control littermates were evaluated for cued fear extinction. Immobility was increased in <i>Dup/+</i> compared to control while it was decreased in the <i>Del/+</i> carriers during the two test periods for extinction (Ext1 (24 h) and Ext2 (48 h)). After one week the cued fear retrieval was observed in the <i>Dup/+</i> mice compared to their wt littermates whereas it was absent in the <i>Del/+</i> compared to their control. <b>(F-G)</b> Three-chamber sociability test. <b>(F)</b> Exploration time (s) of the first congener in the social interest session (session 1) and total exploration time of familiar and novel congeners in the social discrimination session (session 2). No delay was used between the two sessions. <b>(G)</b> Discrimination index was calculated as the ratio of time spent exploring the novel congener vs the familiar one. Data are represented as the mean ± s.e.m. Cohort used included 18 <i>Del/+</i>, 24 wt, 11 <i>Del/Dup</i>, and 11 <i>Dup/+</i> animals. <i>Post hoc</i> Tukey's and Mann Whitney U tests were performed following significant results in one-way ANOVA or Kruskal Wallis analysis, respectively. *<i>P</i> < 0.05 vs wt, **<i>P</i> < 0.01 vs wt, ***<i>P</i> < 0.001 vs wt, ^# <i>P</i> < 0.05 vs all other groups.</p

General behavioral characterization of <i>Kansl1</i>^<i>+/-</i> cohorts.

<p><b>(A)</b> Body weight (g) of adult animals at 15, 17 and 19 weeks of age with Body length (distance from snout to tail basis) and Body fat percentage measured by qNMR of 20-week-old animals. Compared to wt littermates, <i>Kansl1</i>^+/- animals show body weight, size and adiposity deficits. <b>(B)</b> Circadian activity test. Graphs plot the spontaneous ambulatory activity (count) and the vertical activity/rears (count) during dark and light phases. <b>(C)</b> Open field test. Distance travelled (m), and vertical activity/rears (count) over 30 min of testing. <b>(D)</b> Repetitive behaviors. Graph plots occurences of rearing, jumping, climbing and digging behaviors (count) during 10 min of observation in a novel home cage. <i>Kansl1</i>^<i>+/-</i> animals show increase of rearing and decrease of digging levels reflecting an alteration of exploration activity. (<b>E</b>) Results are expressed as the time (s) that mice remained on an accelerating rod before falling during the training phase over 3 consecutive days of the rotarod test. (<b>F</b>) Corresponding rotational velocity (rpm) at the time of falling during the challenge phase of the rotarod test. The graph plots the time (s) that mice stayed on the rod when tested at constant speeds between 4 and 40 rpm. (<b>D</b>) Four-paw grip test. The conclusion of the rotarod and grip tests is that <i>Kansl1</i>^<i>+/-</i> animals show locomotor coordination improvements without alterations of muscular strenght. Graphs depict mean + s.e.m.. (<b>B-D</b>) Student’s t-test, *<i>P</i> < 0.05, **<i>P</i> < 0.01. (<b>A, E-G</b>) Repeated Measures ANOVA "genotype" analysis, Tukey's test, *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

Differences in whole brain volume and relative volumes (normalized or overall brain volume) for the hippocampus, amygdala, nucleus accumbens, cingulate complex, entorhinal cortex and the frontal region of the 17q21.31 mouse models.

<p>All plots use box-and-whiskers diagrams. The box indicates data that lie within the 25th and 75th percentile. The horizontal line in the box specifies the median of the data, and the whiskers the full range of the data with the individual dots being the outliers. (<b>A</b>) Total brain volume is significantly different between the 4 genotypes at 0.1% FDR. (<b>B</b>) Relative volumes. For each structure, the following data are shown: on the left a bar graph indicating the relative size of the entire structure/region expressed as a percentage of total brain volume. Significance is based on the f-statistic resulting from comparing the 4 genotypes. NS = not significant, * = significant at 1% FDR, ** = significant at 0.1% FDR. In the center, the 3D surface renderings focus on significant changes for the structure of interest only. In grey the surface of the entire brain, in yellow the surface of the structure/region of interest and in purple all areas inside that structure/region that are significantly different between the genotypes at 0.1% FDR. On the right is a coronal slice through the average MR brain image. All colored regions indicate areas where the relative volume is significantly different between the 4 genotypes at 0.1% FDR. Cohort used included 8 <i>Del/+</i>, 10 wt, 11 <i>Del/Dup</i>, and 8 <i>Dup/+</i> animals.</p

Learning and memory phenotypes in the <i>Kansl1</i>^<i>+/-</i>.

<p><b>(A)</b> Novel object recognition test. Discrimination index was calculated as the ratio of time spent exploring the novel object vs the familiar object in the choice trial after a 3 h retention delay. <b>(B)</b> Fear conditioning test. Plots represent the fraction of time spent freezing during test sessions. As above the 6-min context session was run 24 h after conditioning and the 8-min cue session was performed 5h after the context. A sequence of 2-min with no cue and 2 min with light/auditory conditioning stimulus was repeated two times. <b>(C-D)</b> Three-chamber sociability test. <b>(C)</b> Exploration time (s) of the first congener in the social interest session (session 1) and total exploration time of familiar and novel congeners in the social discrimination session (session 2). No delay was used between the two sessions. <b>(D)</b> Discrimination index was calculated as the ratio of time spent exploring the novel congener vs the familiar one. Data are represented as the mean ± s.e.m.. Cohort used included 8 <i>Kansl1</i>^<i>+/-</i> and 10 wt animals. Tukey's and Mann Whitney U tests following a significant one-way ANOVA and Kruskal Wallis analysis, respectively. *<i>P</i> < 0.05 vs wt, **<i>P</i> < 0.01 vs wt, ***<i>P</i> < 0.001 vs wt, ^# <i>P</i> < 0.05 vs all other groups.</p