Mean permeation rates and efflux ratios in Caco-2 cell monolayers.

<p>Data are the mean of three independent determinations (samples in triplicate) each with SEM <10%. A: apical; B: basolateral.</p

GNTI enhances maximal Ca²⁺ mobilization by noradrenaline at α_1A-AR without affecting potency (A); maximal PI hydrolysis is not increased (B).

<p>Some intermediate curves have been omitted for clarity. Error bars represent mean ± S.E.M. For raw data, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070701#pone.0070701.s001" target="_blank">Datasets S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070701#pone.0070701.s002" target="_blank">S2</a>.</p

Structural similarities between GNTI and α₁-AR ligands.

<p>Structural similarities between GNTI and α₁-AR ligands.</p

Antagonism of N/OFQ at NOP by JDTic (A) and SB-612,111 (B): inhibition of cAMP production.

<p>Error bars represent mean ± S.E.M. For raw data, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070701#pone.0070701.s005" target="_blank">Dataset S5</a>.</p

GNTI is a weak antagonist of acetylcholine at M₁-R (A); JDTic weakly inhibits the noradrenaline transporter (B).

<p>Error bars represent mean ± S.E.M. For raw data, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070701#pone.0070701.s003" target="_blank">Datasets S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070701#pone.0070701.s004" target="_blank">S4</a>.</p

Binding affinities of nor-BNI, GNTI and JDTic for 46 neurotransmitter receptors and transporters, determined by radioligand displacement.

<p>Submicromolar affinities are shown in bold; blank cells indicate <i>K</i>_i≥10 µM. For details (uncertainty, radioligand, membrane type, species), see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070701#pone.0070701.s007" target="_blank">Table S1</a>. For binding curves, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070701#pone.0070701.s006" target="_blank">File S1</a>.</p

Forced swimming and locomotor activity behaviors 28 days after gene transfer.

<p>(<b>a</b>) During the first exposure to the FST, there were no differences in immobility (IM; <i>F</i>_2,41 = 1.20, not significant), swimming (SW; <i>F</i>_2,41 = 1.60, not significant) or climbing (CL; <i>F</i>_2,41 = 0.05, not significant) behaviors (mean ± SEM) among rats given the dnSPRY2, LacZ, or wtSPRY2 vector into the dorsal HIP. (<b>b</b>) Differences in immobility (<i>F</i>_2,41 = 5.77, <i>P</i><0.01) and swimming (<i>F</i>_2,41 = 6.18, <i>P</i><0.01) emerged upon re-exposure to forced swimming 24 hr later. (<b>c</b>) Vectors did not affect activity in an open field (<i>F</i>_22,451 = 0.09, not significant). *<i>P</i><0.05 compared to LacZ, ^^<i>P</i><0.01 compared to dnSPRY2, Fisher’s protected <i>t</i>-tests, 14–15 rats/group.</p

Effect of viral-mediated gene transfer in the dorsal HIP.

<p>(<b>a</b>) Detection of dnSPRY2 (tagged with a-flag epitope) 3 days after gene transfer (scale bars: 200 μm or [<i>inset</i>] 100 μm). (<b>b</b>) Net number (mean ± SEM) of BrdU-labeled cells (per 40 μm section) 28 days after gene transfer, expressed as the difference between the hemisphere treated with HSV-dnSPRY2, HSV-LacZ, or HSV-wtSPRY2 and the contralateral hemisphere treated with HSV-LacZ (<i>F</i>_2,28 = 7.27, <i>P</i><0.01). (<b>c</b>) Viral vector treatments did not differentially affect the percentage (mean ± SEM) of cells expressing neuronal (NeuN; <i>F</i>_2,28 = 1.99, not significant), glial (S100ß; <i>F</i>_2,28 = 0.98, not significant) or undefined (other; <i>F</i>_2,28 = 1.57, not significant) cell markers. (<b>d</b>) Brightfield microscopy showing BrdU-labeled cells after HSV-dnSPRY2 or (<b>e</b>) HSV-LacZ. (<b>f</b>) Confocal microscopy showing a BrdU-labeled cell (red), (<b>g</b>) NeuN-labeled neurons (green), and (<b>h</b>) a double-labeled neuron (<i>overlay</i>, yellow; scale bar: 10 μm). (<b>i</b>) BrdU-labeled cells (red) were occasionally double-labeled with S100ß (<b>j</b>, blue) indicating glial cells (<b>k</b>, <i>overlay</i>, violet; scale bar: 10 μm). *<i>P</i><0.05 compared to LacZ, ^^<i>P</i><0.01 compared to dnSPRY2, Fisher’s protected <i>t</i>-tests, 9–11 rats/group.</p

Highly simplified schematic depicting a potential pathway by which disruption of SPRY2 function produces elevated neurogenesis and antidepressant-like behaviors.

<p>Binding of growth factors (GF) such as FGF2 at RTKs activates ERK-dependent signaling cascades (green arrows), resulting in processes that tend to increase neurogenesis and produce antidepressant-like behaviors. However, this process also results in elevated SPRY2 transcription and activation (phosphorylation), which tends to inhibit these processes. Electroconvulsive seizure (ECS) activates GFs and inhibits SPRY2, both of which promote neurogenesis and antidepressant-like behaviors.</p

Fear and anxiety-like behaviors 28 days after gene transfer.

<p>(<b>a</b>) During the first test session, there were no differences in the time course (<i>F</i>_4,40 = 0.71, not significant) or overall mean (<i>inset</i>: <i>F</i>_2,20 = 0.13, not significant) of FPS (mean ± SEM). (<b>b</b>) Upon re-testing 48 hr later, there were differential effects of the viral vectors in the time course (<i>F</i>_4,40 = 2.63, <i>P</i><0.05) and overall mean (<i>inset</i>: <i>F</i>_2,20 = 7.61, <i>P</i><0.01) of FPS. (<b>c</b>) In separate rats, there were no differences in time spent on the open arms of the EPM (<i>F</i>_2,20 = 0.34, not significant), *<i>P</i><0.05, **<i>P</i><0.01 compared to LacZ at the 10-min time point, ^^<i>P</i><0.01 compared to wtSPRY2 at the 10-min time point, Fisher’s protected <i>t</i>-tests, 7–8 rats/group.</p