<i>Trib3</i> is upregulated in the mouse anterior piriform cortex in response to leucine-deficient diet.

<p>Adult wild type mice consumed either a diet lacking leucine (Leu−; n = 5) or a corresponding control diet containing leucine (Leu+; n = 5), and, after 6 h of feeding, <i>Trib3</i> expression in the indicated brain regions was quantified by RT-qPCR. The results are presented as the mean ± SEM, and expressed relative to the level of <i>Trib3</i> mRNA in the anterior piriform cortex of the control diet (Leu+) group. *<i>P</i><0.05 comparing leucine-starved and control diet-fed groups.</p

Rejection of amino acid-imbalanced diet in mice is not influenced by the deletion of <i>Trib3</i>.

<p>(<b>A</b>) Consumption of a diet lacking the essential amino acid leucine (Leu−), compared to the consumption of a corresponding nutritionally complete control diet (Leu+). Adult <i>Trib3</i>^+/+ and <i>Trib3</i>^−/− (n = 7 per genotype) mice were trained with food deprivation during the dark phase, and, during the light phase, food intake was measured for each animal at the indicated time-points by weighing the remaining food. For each animal, the intake of Leu− diet was compared to that of the Leu+ diet, and the average difference in the consumption of the Leu− diet relative to the Leu+ diet is expressed in percent ± SEM for each genotype. (<b>B</b>) Body weight and (<b>C</b>) body weight loss due to overnight food deprivation do not differ between <i>Trib3</i>-deficient and wild type adult mice. For B and C, four-month-old group-housed <i>Trib3</i>^+/+ (n = 5 for both males and females) and <i>Trib3</i>^−/− (n = 8 for both males and females) mice were maintained on the Leu+ diet <i>ad libitum</i> for two weeks to determine their diet-habituated body weight, followed by a single iteration of overnight fasting. The data in B and C are presented as the group means ± SEM.</p

Long-term spatial memory and reversal learning ability in the Morris water maze is not altered in <i>Trib3</i>-deficient mice.

<p>Data are means ± SEM from littermate <i>Trib3</i>^−/− (n = 16) and <i>Trib3</i>^+/+ (n = 10) mice. (A) Escape latencies during four days of hidden-platform training performed at four trials per day. (B) Pool quadrant occupancy in a probe trial performed 24 h after the completion of training. The submerged platform was removed from the pool and the swim trajectory of mice was monitored for 1 min. The pool quadrant that previously contained the platform is designated the target quadrant, and the time spent in each quadrant of the pool is presented as percent of the total search time. (C) Reversal training escape latencies during two days of training performed at four trials per day. For reversal training, the hidden platform was repositioned to the pool quadrant opposite of the initial platform location. (D) Results of a probe trial performed 24 h after reversal training. The probe trial was carried out as in B. The reversal target denotes the pool quadrant that contained the platform during the reversal trainings trials. (E) Swimming speed at different stages of the experiment. The mean speed ± SEM from 60-second swimming sessions with no platform are shown. The habituation session was performed one day before the start of training. For <i>Trib3</i>^+/+, n = 5 per sex, and for <i>Trib3</i>^−/−, n = 8 per sex.</p

<i>Trib3</i> is developmentally regulated in the mouse brain.

<p>(<b>A</b>) RT-qPCR quantification of <i>Trib3</i> expression in wild type C57BL/6J mouse brains at embryonic day (E) 14, 15, 17 and 18, and at postnatal day (P) 0, 2 and 4. The mean <i>Trib3</i> expression level ± SEM at the indicated age is presented relative to the level of <i>Trib3</i> expression at E14 (n = 7 for E17, E18 and P0, n = 6 for E15, n = 5 for E14 and P2, and n = 3 for P4). Means marked with the same letter are not significantly different at the 5% significance level. (<b>B</b>) Lack of <i>Trib3</i> does not lead to altered expression of other <i>Tribbles</i> family genes in the P3 mouse brain. RT-qPCR was used to determine the level of <i>Trib1</i> and <i>Trib2</i> mRNA expression in littermate <i>Trib3</i>^+/+, <i>Trib3</i>^+/− and <i>Trib3</i>^−/− mice (n = 5 per genotype). For both genes, the mean ± SEM is presented relative to the level of expression in <i>Trib3</i>^+/+ mice.</p

Distribution of Wfs1, D₁, and D₅ proteins in selected regions of the adult mouse brain.

<p>Distribution of Wfs1, D1, and D5 proteins in selected regions of the adult mouse brain. Immunohistochemistry on coronal sections. The detected proteins are indicated above. In the cortex Wfs1, D1, and D5 are all present in layer I, upper part of layer II/III, and in layer V (images show somatosensory cortex; A-C); in the hippocampus Wfs1, D1, and D5 are simultaneously present in pyr and slm of CA1 (D-F); Wfs1, D1 and D5 are all present in Rt and Sth; in amygdala Wfs1 is strongly present in CeA, the lateral edge of BL, and medial and cortical nuclei (G-I), whereas D1 and D5 show only weak signal in CeA and BL (G-I); in the dorsolateral thalamus Wfs1, D1, and D5 are delineating dLG and VPM, note that strongly Wfs1-positive fibers are present in fi, but no D1 or D5 is seen there (J-L), in the substantia nigra Wfs1 has similar distribution with D1, but not with D5 (M-O). For abbreviations, see list. Scale bar is 100 μm in A-C, 1 mm in D-L, 500 μm in M-O.</p

Binding of D₁/D₅ specific ligand [³H]SCH23390 to hippocampal membranes of wt and <i>Wfs1</i> knockout mice.

<p>Comparison of specific binding of radioligand [³H]SCH23390 to hippocampal membranes of wt and <i>Wfs1</i> knockout mice. (A) Binding curve of [³H]SCH23390 binding to pooled samples of wt (triangle) and <i>Wfs1</i> knockout (circle) mice. The membrane suspensions (3 mg/well) were incubated with different concentrations of [³H]SCH23390 for 60 min and bound radioactivity was measured. Data are presented as mean ± SEM from experiments (n = 3) performed in duplicates. (B) The level of [³H]SCH23390 binding sites of individual wt and <i>Wfs1</i> knockout mice determined in hippocampal membrane suspensions (6.7 mg/ml.) incubated with 4 nM radioligand. Data presented as mean ± SEM of all the mice tested. *P < 0.05. Data of individual mice are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172825#pone.0172825.s006" target="_blank">S1 Table</a>.</p

The expression of <i>Wfs1</i> and <i>Drd1a</i> in the adult red-eared slider turtle (<i>Trachemys scripta</i>) brain.

<p>The expression of <i>Wfs1</i> and <i>Drd1a</i> in the adult red-eared slider turtle (<i>Trachemys scripta</i>) brain, shown by mRNA <i>in situ</i> hybridization on coronal brain sections. The sections are in anterio-posterior order from left to right. The probes are indicated on the left side of the figure. <i>Wfs1</i> expression is widespread in the brain of <i>T</i>.<i>scripta</i>, being distinguishedly strong in MC, DC, PT and near the ventricular surface in the caudal DVR (A-D). <i>Drd1a</i> expression occupies the same regions as that of <i>Wfs1</i>, but is missing in MC and very weak in DC and caudal DVR (E-H). Unlike <i>Wfs1</i>, <i>Drd1a</i> is present in LC (E-G). For abbreviations, see list. Scale bar is 1 mm.</p

The expression of <i>Wfs1</i> and <i>Drd1a</i> in the adult chick brain.

<p>The expression of <i>Wfs1</i> and <i>Drd1a</i> in the adult chick brain, shown by mRNA <i>in situ</i> hybridization on coronal brain sections. The section plane is shown on image L. The probes are indicated on the left side of the figure. Both, <i>Wfs1</i> and <i>Drd1a</i> show strong expression in rostral to medial MSt (A-F). In Acb and StPalAcb, the expression of <i>Wfs1</i> is substantially weaker than in the surrounding striatal structures (A-B). The expression of <i>Drd1a</i> is weak in Acb and StPalAcb, and is missing in SPO (D-F). In LSt, both <i>Wfs1</i> and <i>Drd1a</i> expression show strengthening gradient in rostrocaudal direction (A-G,J). <i>Wfs1</i>-expressing cells in PHi are shown in higher magnification (I). In the adult brain, ADo and APir are delineated with <i>Wfs1</i> expression, but remain hardly distinguishable by <i>Drd1a</i> expression (H,K). Note that GP is devoid of both <i>Drd1a</i> and <i>Wfs1</i> (B,E,G,J). For abbreviations, see list. Scale bar is 1 mm in A-H and J-K and 500 μm in I.</p

Distribution of Wfs1 protein in the striata of common quail (<i>Coturnix coturnix</i>) and red-eared slider turtle (<i>Trachemys scripta</i>) brains.

<p>Distribution of Wfs1 protein in the striata of quail (<i>Coturnix coturnix</i>) and red-eared slider turtle (<i>Trachemys scripta</i>) brains. The panel shows fluorescent immunohistochemistry on coronal brain sections. Wfs1 expression (green) is seen in medial striatum of quail (A, MSt) and turtle (D, St). Concave arrowheads point the expression in soma in both species (C, F). Wfs1 is detectable in neuronal processes of quail (C, concave arrowheads). Nuclei are counterstained with DAPI (blue). Scale bar is 100 μm.</p

Wfs1 is expressed in dopaminoceptive regions of the amniote brain and modulates levels of D1-like receptors - Sheet 0

<p>Wfs1 is expressed in dopaminoceptive regions of the amniote brain and modulates levels of D1-like receptors</p> - Sheet