Acute stress induces miR-135a and miR-124 downregulation in the amygdala.

<p>(A) The differential expression of mature miRNAs in the amygdala was evaluated by microarray analysis after 2 hours of restraint. The MA plot shows relative change values, expressed as log₂ ratio (stress vs control), plotted against average log intensity ((log₂Hy5+log₂Hy3)/2). (B) Levels of mature miRNAs are quantified in the amygdala RNA pool by qRT-PCR, using U6B as internal control. The statistical test used for comparison was one-way ANOVA (<i>n</i>=9). Values are means ± SE *<i>P</i> <0.001 versus naive control mice.</p

miR-135a and miR-124 regulate the expression of the endogenous MR protein.

<p>(A) N2a cells were transfected with miR-135a (p135a) and miR-124(p124) overexpressing vectors as indicated. p135a+p124 corresponds to the transfection with an equimolar mixture of the two vectors. A representative blot is shown. (B) Western blot analysis of endogenous MR expression was quantified by densitometry, normalized to actin as loading control and expressed relatively to empty vector transfected cells. Data represent the mean from three biological samples and three technical replicates ± SE. *<i>P</i> < 0.05, <i>**P</i> < 0.05 (pairwise Student’s <i>t</i>-test). (C) Cerebellar granule neurons (6+4 DIV) were transfected with LNA antisense oligonucleotides or scramble LNA as negative control. A representative blot is shown. (D) Western blot analysis of endogenous MR expression was quantified by densitometry, normalized to tubulin and expressed relatively to scramble LNA transfected cells. Data represent the mean from four biological samples and three technical replicates ± SE. *<i>P</i> < 0.05, (pairwise Student’s <i>t</i>-test).</p

Acute stress increases MR protein levels in amygdala.

<p>(A) Steady state MR protein levels were measured by western blot analysis immediately after 2 hours of restraint. Lysates were obtained from pooled amygdala nuclei (<i>n</i>=4), 5 pools were generated from naive (<i>n</i>=20) and restrained mice (<i>n</i>=20). MR expression was normalized to actin signals in the same blot. Quantitative values are shown as mean ± SE <i>*P</i> < 0.05 (pairwise Student’s <i>t</i>-test). (B) qRT-PCR analysis of Nr3c2 transcript levels in naive and stressed mice (<i>n</i>=12 for both groups). Data are presented as mean ± SE. Ex 6-7 and exs 8-9 refer to the amplicons studied, corresponding to exons 6 and 7 (exs 6-7), and to exons 8 and 9 (exs 8-9) of the Nr3c2 coding sequence.</p

Nr3c2 reporter is regulated by miR-135a.

<p>(A) High probability miRNA target sequences in the mouse Nr3c2 3’ UTR are drawn. Candidate miRNAs are predicted by microT v 3.0, TargetScan 5.2, and PicTar algoritms. Positions of mir-135a and miR-124 target sequences in the mouse annotated Nr3c2 3’ UTR and details of miRNA/mRNA base pairing are indicated. Beneath miRNA sequences, nucleotides mutated at the level of miRNA binding sites in the mutant constructs Nr3c2 m135a (mutated at both miR-135a seed binding sites) and Nr3c2 m124 (mutated at both miR-124 seed binding sites), are indicated (nts in bold). (B) Nr3c2 luciferase reporter (Nr3c2), and mutant reporter constructs were co-transfected into Hela cells together with empty vector or miRNA expression vectors (p135a and p124). Luciferase activity was measured 24 hours post-transfection. Values are expressed relatively to the internal renilla luciferase activity and presented as percentage of the activity achieved in the presence of the empty control vector. Results are shown as means ± SE (n=6). **<i>P</i><0.005 (pairwise Student’s <i>t</i>-test).</p

Nicotine microperfusion impairs VOR gain adaptation.

<p>The flocculus of the mouse was located using extracellular electrophysiological recordings during visual stimulation. (<b>A</b>) Increased complex spike activity during horizontal, contralateral movement of the visual field confirmed the location of vertical axis (VA) Purkinje cells. Based on the polarity of the waveform of Purkinje cell activity identified in each track, the location of the granule cell layer was determined, and a dye-labelled vehicle solution with or without 5–10 ng nicotine (n = 8 and n = 7, respectively) was injected. (<b>B</b>) Dye diffusion was analysed histologically and compared with the unfolded (according to points a-d) mouse floccular map <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064828#pone.0064828-Schonewille2" target="_blank">[72]</a>. Only injections covering at least 10% of the VA were included. The effect on motor learning was assessed by subjecting mice, 15 min after injection, to five 10 min sessions of in-phase vestibular and visual input, aimed at decreasing the VOR gain. (<b>C</b>) Mice injected with nicotine showed a significantly impaired ability to decrease their VOR gain. Nicotine did not affect the timing (phase) of the VOR, and the gain during the training was also not affected, indicating that the deficit is specific for adaptation rather than performance.</p

α7-nAChR stimulation facilitates LTP <i>in vivo</i>.

<p>LFP recordings were performed from the granular layer of the Crus-IIa of the right cerebellar hemisphere in response to air-puff stimulation of the homolateral whisker pad. A TSS air-puff induction pattern was delivered at the end of a 5 min microperfusion of drugs in different combinations: TSS+Krebs’ solution (n = 7), just 50 µM nicotine (n = 6), TSS +50 µM nicotine (n = 6), TSS +100 mM choline (n = 5), TSS +50 µM nicotine +0.5 µM MLA (n = 5). <i>Left</i>, LFPs recorded before and after TSS+nicotine (average of 100 traces). <i>Right</i>, time course of the LFP amplitude changes (mean±SEM). Drug microperfusion is indicated by a bar and TSS by an arrow.</p

Postsynaptic induction of nicotine facilitated LTP through intracellular Ca²⁺ regulation.

<p>Intracellular Ca²⁺ concentration, [Ca²⁺]_i, was measured in granule cell dendrites as OG1 relative fluorescence, ΔF/F₀. Choline 10 mM was applied for 100 seconds just before a 10-pulse (100 Hz) mossy fibre burst, while holding the granule cell at –40 mV. <b>(A)</b> The sequence of pseudocolour images show higher Ca²⁺ increase in a granule cell dendritic ending when choline is perfused than in control recordings. <b>(B)</b> Time course of ΔF/F₀ in recordings obtained in control and following choline perfusion (<i>left</i>) and relative EPSC changes (taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064828#pone-0064828-g002" target="_blank">Fig. 2</a>) as a function of maximum ΔF/F₀ during bursts of different duration (<i>right</i>). Choline moves the point (mean±SEM, n = 5) corresponding to the 10-pulse burst from LTD to LTP (arrow).</p

Evidence for presynaptic expression of nicotine facilitated LTP.

<p>Percent changes induced by 10-pulse bursts in EPSC amplitude, release probability (<i>p</i>), coefficient of variation (<i>CV</i>), and failure rate (<i>FR</i>) between 15 and 20 min after the induction of plasticity in the experiments A-C of Fig. 2 (p<0.01 for all parameters).</p

α7-nAChR activation at the mossy fibre-granule cell synapse.

<p>In A, B, and C, patch-clamp recordings performed from granule cells voltage-clamped at –70 mV in cerebellar slices. Drugs were applied for 100 seconds (black bar). (<b>A</b>) EPSC amplitude changes caused by application of nicotinic agents. (<i>Left)</i> Average traces of 10 contiguous EPSCs taken from a representative experiment (1 µM nicotine). <i>(Right)</i> Time course of EPSC amplitude changes (mean ± SEM) during application of 1 µM nicotine (n = 56), 10 mM choline (n = 5), 1 µM nicotine +10 nM MLA (n = 4), 50 nM epibatidine (n = 4), 10 nM MLA (n = 5), 1µM DHβE (n = 7). (<b>B</b>) EPSC amplitude changes in C57/BL6 (n = 4) and α7-nAChR KO mice (n = 4) during application of 1 µM nicotine. Same panel layout as in A. (<b>C</b>) The effect of postsynaptic calcium buffering (0.1 mM or 10 mM intracellular BAPTA) on the action of 1 µM nicotine (black bar). <i>(Left)</i> Average traces from 10 contiguous AMPA-EPSCs and NMDA-EPSCs (isolated with 10 µM NBQX in Mg²⁺-free medium) in representative recordings. <i>(Right)</i> Ensemble effects on AMPA-EPSC PPR and NMDA-EPSC amplitude (mean ± SEM). (<b>D</b>) Immunolabelling for α7-nAChR subunit in electron micrographs at the mossy fibre-granule cell synapse. An immunopositive pre-terminal mossy fibre (mf, bordered by arrows) opens up in a large bouton, which surrounds granule cell dendrites (<i>d</i>) (scale bar 0.5 µm). The inset shows a granule cell dendrite (<i>d</i>), contacted by a mossy fibre bouton (<i>b</i>), bearing an immunopositive post-synaptic specialisation (arrow). Scale bar 0.8 µm.</p