Time course of Dtx:UII-induced pedunculopontine tegmental nucleus cholinergic cell loss.

<p><b>Panels A-C</b>: Representative sagittal PPTg sections under bright field microscopy showing choline acetyltransferase (ChAT) immunoreactivity (ChAT-IR; brown cells), 1 (A), 4 (B), and 7 (C) days following unilateral PPTg infusion of Dtx:UII (right panels) and vehicle (0.01 M PBS) infusion into the contralateral PPTg (left panels). Dtx:UII infusion induced a slight loss of PPTg ChAT-IR cells by 4 days post-infusion that became extensive by 7 days post-infusion. <b>Panel D:</b> Numbers of ChAT-IR positive cells counted at each of five time points (1, 4, 7, 14, and 28 days, n = 3 per time point) following unilateral PPTg infusion of Dtx:UII (black bars) and vehicle infusion into the contralateral PPTg (white bars) hemispheres. Dtx:UII infusion significantly reduced the number of ChAT-IR cells by 4 days post-infusion. Error bars represent ± SEM.</p

Intravenous self-administration of cocaine is not altered by bilateral PPTg cholinergic cell loss.

<p>Bilateral PPTg cholinergic cell lesions (solid circles; n = 5) did not significantly affect cocaine self-administration relative to sham lesions (open squares; n = 5), either in terms of the number of cocaine infusions in daily sessions (top) or the number of active lever presses to attain those infusions (bottom panel). All rats showed a decrease in cocaine intake on the first post-lesion testing day that returned to pre-lesion baseline levels by the third post-lesion testing day. The ability to distinguish between the retractable and stationary levers was not compromised by PPTg cholinergic cell loss. Error bars represent ± SEM.</p

Lesions induced uniform cholinergic cell loss throughout the rostro-caudal extent of the pedunculopontine tegmental nucleus (PPTg).

<p><b>Panels A–B</b>: Low magnification (4X) coronal PPTg sections (A-P -8.3) under bright field microscopy showing ChAT immunoreactive neurons (ChAT-IR; brown cells) following bilateral PPTg infusion of Dtx:UII (B) or vehicle (0.01 M PBS; A) from rats used in behavioral experiments. For better visualization of ChAT immunoreactivity, delimited PPTg areas in (A) and (B) are shown at higher magnification (20X) in A1 and A2 and B1 and B2, respectively. Similarly, delimited LDTg areas in (A) and (B) are shown in A3 and A4 and B3 and B4, respectively. <b>Panel C:</b> Numbers of ChAT-IR cells counted in each of two hemispheres at each of three rostro-caudal PPTg levels (A/P -8.3 caudal, A/P −7.7 medial and A/P −6.8 rostral) following cholinergic cell lesions (white bars; n = 14) or sham lesions (black bars; n = 13). Cholinergic cell loss was confined to the PPTg, as the number of ChAT immunoreactive neurons cells in the LDTg was not significantly changed (panel C, inset). Error bars represent ± SEM.</p

Dtx::UII infusion induced loss of PPTg glutamatergic and GABAergic cells.

<p><b>Panels A and B:</b> Representative sagittal PPTg sections showing ChAT immunoreactive neurons (ChAT-IR; brown cells) and GAD_65/67 mRNA (purple cells) under bright field microscopy (left) and VGluT2 mRNA as white aggregates under epiluminescence microscopy (right) 14 days following unilateral PPTg infusion of vehicle (0.01 M PBS; A and A′) and Dtx:UII (B and B′) into the contralateral PPTg. <b>Panel C:</b> Numbers of cholinergic (ChAT), glutamatergic (VGluT2), and GABAergic (GAD_65/67) cells following unilateral PPTg infusion of Dtx:UII (left panels), expressed as a percentage of the number of each cell type counted in the contralateral hemisphere following vehicle infusion (n = 3). Unilateral PPTg Dtx::UII infusion induced significant loss of cholinergic, glutamatergic, and GABAergic cells. Error bars represent ± SEM.</p

Cocaine or heroin conditioned place preferences are not altered by bilateral pedunculopontine tegmental nucleus (PPTg) cholinergic cell loss.

<p>For cocaine conditioned place preference (20 mg/kg; i.p.; left) PPTg cholinergic cell-lesioned rats (black bar; n = 4) and sham-lesioned rats (gray bar; n = 4) each showed a significant increase in the amount of time spent in the cocaine-paired chamber as a result of place conditioning. For heroin conditioned place preference (0.5 mg/kg; i.p.; right) PPTg cholinergic cell-lesioned rats (black bar; n = 6) and sham-lesioned (gray bar; n = 5) each showed a significant increase in the amount of time spent in the heroin-paired chamber as a result of place conditioning. Error bars represent ± SEM.</p

Spontaneous, saline-induced, and cocaine-induced locomotion are not altered by bilateral pedunculopontine tegmental nucleus (PPTg) cholinergic cell loss.

<p>Bilateral PPTg cholinergic cell lesions (solid circles; n = 4) did not significantly alter spontaneous locomotion (A), saline-induced locomotion (B), or cocaine-induced locomotion (C) relative to sham lesions (open squares, n = 4). Insets in A–C show total locomotion across the two-hour testing period in each of three conditions (n.s.  =  non significant). Note that locomotion was tested in the same lesioned and control rats used for the cocaine place preference (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084412#pone-0084412-g002" target="_blank">Fig. 2</a>). Error bars represent ± SEM.</p

Intravenous self-administration of heroin is not altered by bilateral pedunculopontine tegmental nucleus (PPTg) cholinergic cell loss.

<p>Bilateral PPTg cholinergic cell lesions (solid circles; n = 4) did not significantly affect heroin self-administration relative to sham lesions (open squares; n = 4), either in terms of the number of heroin infusions in daily sessions (top) or the number of active lever presses to attain those infusions (bottom panel). All rats showed a slight increase in heroin intake across the post-lesion testing period. The ability to distinguish between the retractable heroin and stationary levers was not compromised by PPTg cholinergic cell loss. Error bars represent ± SEM.</p

TGF-β1 Reduces miR-29a Expression to Promote Tumorigenicity and Metastasis of Cholangiocarcinoma by Targeting HDAC4

<div><p>Transforming growth factor β1 (TGF-β1) and miRNAs play important roles in cholangiocarcinoma progression. In this study, miR-29a level was found significantly decreased in both cholangiocarcinoma tissues and tumor cell lines. TGF-β1 reduced miR-29a expression in tumor cell lines. Furthermore, anti-miR-29a reduced the proliferation and metastasis capacity of cholangiocarcinoma cell lines in vitro, overexpression of miR-29a counteracted TGF-β1-mediated cell growth and metastasis. Subsequent investigation identified HDAC4 is a direct target of miR-29a. In addition, restoration of HDAC4 attenuated miR-29a-mediated inhibition of cell proliferation and metastasis. Conclusions: TGF-β1/miR-29a/HDAC4 pathway contributes to the pathogenesis of cholangiocarcinoma and our data provide new therapeutic targets for cholangiocarcinoma.</p></div

Enhanced expression of miR-29a attenuates TGF-β1-mediated cholangio- carcinoma cell metastasis.

<p>Wound healing assay was performed in FRH–0201 and CCLP–1 cells (A-C), 48h after transfection with anti-miR-29a. When the two tumor cell lines were treated with miR-29a mimic, wound healing assay was performed after additional treatment with 5ng ml^-1TGF-β1 for 48h. Data are shown as mean±SD; *p<0.05;**P<0.01;*** P<0.001.</p

Transwell cell invasion assay.

<p>(A and B) Transwell assay was administrated in the two tumor cell lines, 48h after treatment with anti-miR-29a. When the two tumor cell lines were treated with miR-29a mimic, the migrating cells were counted after additional incubation with 5ng ml^-1TGF-β1 for 48h. Data are shown as mean±SD; **P<0.01;*** P<0.001.</p