Detection of Trefoil factor 1 (TFF1) in nigrostriatal projection neurons by Fluorogold (FG) labelling.

<p>Representative photomicrographs of double immunofluorescence stainings for TFF1 and the retrograde tracer FG at the level of substantia nigra pars compacta (SNc) 10 days after intrastriatal FG injection. A subpopulation of TFF1-ir cells was found to co-express FG identifying these as projection neurons (arrows). Scale bars upper panel: 100 µm, lower panel: 50 µm.</p

Co-expression of trefoil factor 1 (TFF1) and calcium-binding proteins in the ventral mesencephalon of adult rats.

<p>Double immunofluorescence stainings for TFF1 and the calcium-binding proteins calretinin (CR), calbindin (CB) or parvalbumin (PV). Note that many TFF1-immunoreactive (-ir) cells co-expressed CR (arrows), but not all CR-ir cells co-localized with TFF1 (arrowheads). Similarly, co-localization with CB was seen for a few TFF1-ir cells (arrows). As expected from the distribution pattern depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076592#pone-0076592-g001" target="_blank">Figure 1</a> no co-localization was detected for TFF1-ir cells with PV (arrows). Scale bars middle panel: 50 µm, lower panel: 100 µm.</p

Co-expression of trefoil factor 1 (TFF1) and tyrosine hydroxylase (TH) in the ventral mesencephalon of adult rats.

<p>Double immunofluorescence staining for TFF1 (red) and TH (green) in the ventral mesencephalon of adult rats. Note that nearly all TFF1-immunoreactive (-ir) cells co-localized with TH, while many TH-ir cells did not co-express TFF1. Scale bars: 1 mm (overview); 100 µm (magnification). Abbreviations: SNc, substantia nigra pars compacta; SNl, substantia nigra pars lateralis; VTA, ventral tegmental area.</p

Quantification of trefoil factor 1 (TFF1) and tyrosine hydroxylase (TH) co-expressing cells in adult rats.

<p>Percentage of trefoil factor 1-immunoreactive (TFF1-ir) cells that co-localized with tyrosine hydroxylase (TH) (A) and percentage of TH-ir cells that co-localized with TFF1 (B) in the substantia nigra (SN) and ventral tegmental area (VTA) of adult rats. *: p<0.05 vs. corresponding VTA values, n = 4.</p

Loss of trefoil factor 1 (TFF1) expressing cells in a rat model of Parkinson’s disease.

<p>Representative photomicrographs of tyrosine hydroxylase (TH; A) and TFF1 (B) in brain sections from adult rat ventral mesencephalon at 4 weeks after unilateral 6-hydroxydopamine (6-OHDA) lesion. The lesion resulted in a distinct loss of TH-ir neurons in right SN (A2) as compared to the contralateral, unlesioned control side (A1). Similarly, a reduction of TFF1-ir cells was detected on the lesioned side (B2) as compared to the intact control side (B1). This loss of TFF1-ir cells is better recognized on the enlarged images (B2). Scale bars: 1 mm (overview), 500 µm (magnification).</p

Quantification of trefoil factor 1 (TFF1) and tyrosine hydroxylase (TH) co-expressing cells during development.

<p>A) Quantification of trefoil factor 1-immunoreactive (TFF1-ir) cells co-expressing tyrosine hydroxylase (TH) and B) TH-ir cells co-expressing TFF1 in the substantia nigra (SN) of postnatal (P) day 7, 14, 21 and adult rats. The percentage of TH/TFF1 co-expressing cells was significantly higher for P7 and P14 rats compared to P21 rats, whereas there was no significant difference between the percentages of TFF1-ir cells co-expressing TH. Data are expressed as mean ± SEM (*: p<0.05, n = 4-7). C) Representative double immunofluorescence images of TFF1-ir and TH-ir cells in SN of P7, P14 and P21 rats (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076592#pone-0076592-g002" target="_blank">Figure 2</a> for adult rats). Scale bar: 200 µm.</p

Trefoil factor 1 (TFF1) expression is restricted to neurons.

<p>Representative photomicrographs of double immunofluorescence stainings for TFF1 and astroglial or neuronal markers in the substantia nigra pars compacta (SNc) of adult rats. No co-localization was found of TFF1-positive cells (arrows) with the astroglial marker glial fibrillary acid protein (GFAP) (arrowheads). Notably, a substantial number of the TFF1-ir cells were demonstrated to co-express the pan neuronal marker NeuN (arrows), while some did not (arrowheads). Scale bar: 50 µm.</p