Generation of the <i>Tph2</i>^<i>flox</i> and <i>Tph2</i>^<i>null</i> alleles.

<p>(<b>a</b>) Diagram showing the targeting strategy used to generate the <i>Tph2</i>^{<i>flox(Neo)</i>} allele and the derivation of both <i>Tph2</i>^<i>flox</i> and <i>Tph2</i>^<i>null</i> alleles after the removal of the <i>PGK-neo</i> cassette via flp mediated recombination and the removal of the third exon of <i>Tph2</i> gene after Cre mediated somatic recombination, respectively. Schematic representation of wild-type <i>Tph2</i> genomic locus, <i>Tph2</i>^{<i>flox(Neo)</i>} targeting vector, <i>Tph2</i>^{<i>flox(Neo)</i>}, <i>Tph2</i>^<i>flox</i> and <i>Tph2</i>^<i>null</i> alleles are reported. (<b>b</b>) Southern blot analysis confirming the correct integration of <i>Tph2</i>^{<i>flox(Neo)</i>} targeting vector within the genome of ES cells via homologous recombination. Genomic DNA was digested with EcoRI and EcoRV, or SpeI restriction enzymes and hybridized with a probe external to the left homology arm (probe A) or to the right homology arm (probe B), respectively. Probe A and probe B are indicated in <b>a</b>. (<b>c</b>) PCR genotyping of mice using tail biopsy allowing to discriminate wild-type (<i>Tph2</i>^<i>+/+</i>), <i>Tph2</i>^{<i>flox/+</i>}, <i>Tph2</i>^{<i>flox/flox</i>}, as well as <i>Tph2</i>^{<i>null/+</i>} and <i>Tph2</i>^{<i>null/null</i>} animals. EV: EcoRV; EI: EcoRI; S: SpeI; wt: wild-type; L: ladder.</p

<i>Pet1₂₁₀-Cre</i> transgenic mouse line promotes somatic recombination in a non-serotonergic cell population within the <i>raphe</i> nuclei.

<p>Representative low (<b>a</b>, <b>b</b>, <b>c</b>, <b>d</b>, <b>e</b>) and high (<b>a’-a’’’</b>, <b>b’-b’’’</b>, <b>c’-c’’’</b>, <b>d’-d’’’</b>, <b>e’-e’’’</b>) magnification confocal images of coronal sections of adult <i>Pet1₂₁₀-Cre/ROSA26YFP</i> brains, showing the distribution of YFP (green) and 5-HT (red) immunoreactivity within B8–B9 (<b>a-a’’’</b>), B8 (<b>b-b’’’</b>), B7 (<b>c-c’’’</b>), B6 (<b>d-d’’’</b>), and B1–B3 (<b>e-e’’’</b>) serotonergic nuclei. Boxes highlight the region of each <i>raphe</i> nucleus shown at higher magnification. Cells immunoreactive for both YFP and 5-HT (YFP⁺/5-HT⁺, arrowheads) or exclusively YFP but not 5-HT (YFP⁺/5-HT⁻, arrows) were detected along the antero-posterior extent of the <i>raphe</i> highlighting variable representativeness among the different nuclei. Scale bar: 200 µm (<b>a–e</b>), 30 µm (<b>a’-a’’’</b>, <b>b’-b’’’</b>, <b>c’-c’’’</b>, <b>d’-d’’’</b>, <b>e’-e’’’</b>).</p

Characterization of <i>Tph2</i>^<i>null</i> transcripts.

<p>(<b>a</b>) Schematic representation of RT-PCR products obtained from <i>Tph2</i>^<i>+/+</i>, <i>Tph2Δ3</i> and <i>Tph2Δ3Δ4</i> transcripts using the following primer sets: Fw1 –Rev2, Fw1 –Rev 3, Fw1 –Rev 4, Fw1 –Rev5, Fw1 –Rev6 and Fw1 –Rev11. (<b>b</b>) Agarose 1.5% gel showing the RT-PCR products obtained from <i>Tph2</i>^<i>+/+</i>, <i>Tph2</i>^{<i>null/null</i>} and tamoxifen-treated <i>Tph2</i>^{<i>flox/null</i>}::<i>CMV-CreER</i>^<i>T</i> cDNA using the primer sets described in (<b>a</b>). Two distinct PCR products are obtained using Fw1 –Rev 4 (exon1-4), Fw1 –Rev 5 (exon1-5), Fw1 –Rev 6 (exon1-6), and the Fw1 –Rev 11 (exon1-11) primer sets from cDNA of <i>Tph2</i>^{<i>null/null</i>} and the tamoxifen treated <i>Tph2</i>^{<i>flox/null</i>}::<i>CMV-CreER</i>^<i>T</i> mice. An additional amplicon corresponding in size to the wild-type PCR product is amplified from <i>Tph2</i>^{<i>flox/null</i>}::<i>CMV-CreER</i>^<i>T</i> cDNA. (<b>c</b>) Electropherograms showing the nucleotide sequence obtained sequencing the two distinct amplicons obtained from <i>Tph2</i>^{<i>null/null</i>} cDNA using Fw1—Rev5 primers. Sequencing analysis shows that the second exon of the <i>Tph2</i> transcript is joined to the fourth exon in the <i>Tph2Δ3</i> cDNA, while it is directly connected to the fifth exon in the <i>Tph2Δ3Δ4</i> transcript. L: ladder; N: cDNA from the <i>raphe</i> of a <i>Tph2</i>^{<i>null/null</i>} mice; T: cDNA from the <i>raphe</i> of a <i>Tph2</i>^{<i>flox/null</i>}::<i>CMV-CreER</i>^<i>T</i> tamoxifen-treated mouse sacrificed seven days after the end of the treatment.</p

<i>Pet1₂₁₀-Cre</i> mouse line drives Cre recombinase activity in the serotonergic system.

<p>(<b>a</b>) Dorsal view of a cleared X-gal stained <i>Pet1₂₁₀-Cre/ROSA26R</i> embryo at 11.5 dpc showing that Cre-mediated somatic recombination has occurred in the rostral <i>raphe</i> (arrow). (<b>b</b>) Sagittal section of 12.5 dpc <i>Pet1₂₁₀-Cre/ROSA26R</i> embryo highlighting the presence of the reporter in both the rostral (arrows) and caudal (arrowheads) clusters of developing serotonergic neurons. (<b>c</b>) In P1 <i>Pet1₂₁₀-Cre/ROSA26R</i> brains X-gal staining highlights serotonergic neurons migrated towards their terminal locations within the rhombencephalon. (<b>d–g, d’–g’</b>) Representative coronal sections throughout the antero-posterior extent of the <i>raphe</i> of an adult (P45) <i>Pet1₂₁₀-Cre/ROSA26R</i> brain showing Cre-mediated recombination specifically occurred in all serotonergic nuclei, namely B8–B9 (<b>d, d’</b>), B7 (<b>e, e’</b>), B5–B6 (<b>f, f’</b>) and B1–B3 (<b>g, g’</b>). (<b>h-h’’</b>, <b>i-i’’</b>) no β-galactosidase staining is detectable in anterior brain regions such as cortex (<b>h-h’</b>), hippocampus (<b>h, h’’</b>), substantia nigra (<b>i-i’</b>) and thalamus (<b>i, i’’</b>). Scale bar: 1 mm (<b>a</b>, <b>d–i</b>), 900 µm (<b>c</b>), 600 µm (<b>b</b>), 300 µm (<b>d’</b>-<b>i’</b>, <b>h’’</b>, <b>i’’</b>).</p

<i>Tph2</i>^{<i>null/null</i>} mice are depleted of brain serotonin.

<p>Representative confocal images of P0 wild-type (<b>a</b>, <b>d</b>), <i>Tph2</i>^{<i>flox/flox</i>} (<b>b</b>, <b>e</b>) and <i>Tph2</i>^{<i>null/null</i>} (<b>c</b>, <b>f</b>) brain coronal sections, showing the distribution of 5-HT immunoreactive neurons within B7 (<b>a</b>-<b>c</b>) and B1-B3 (<b>d</b>-<b>f</b>) <i>raphe</i> nuclei. While no differences are visible in the distribution of serotonin immunoractive neurons between wild-type and <i>Tph2</i>^{<i>flox/flox</i>} pups (<b>a</b>-<b>b, d</b>-<b>e</b>), the brain of <i>Tph2</i>^{<i>null/null</i>} pups appear to be devoid of 5-HT throughout its anterior-posterior axis (<b>c</b>, <b>f</b>). Scale bar: 400 μm.</p

Tamoxifen-induced somatic recombination results in a rapid depletion of brain serotonin in adult mice.

<p>(<b>a</b>) Experimental design: <i>Tph2</i>^{<i>flox/null</i>}::<i>CMV-CreER</i>^<i>T</i> mice received tamoxifen injection once per day starting from P60 for 5 consecutive days and sacrificed 1 (D1), 3 (D3), 7 (D7), 30 (D30), 60 (D60) or 90 (D90) days after the end of the treatment, respectively. (<b>b</b>) Representative coronal section of B8-B9 and B7 <i>raphe</i> nuclei showing serotonin immunoreactivity in (from right to left) adult wild-type mice, vehicle-treated <i>Tph2</i>^{<i>flox/null</i>}::<i>CMV-CreER</i>^<i>T</i> control mice sacrificed 30 days after the last injection, tamoxifen-treated <i>Tph2</i>^{<i>flox/null</i>}::<i>CMV-CreER</i>^<i>T</i> mice sacrificed 1, 3, 7 and 30 days after the end of treatment, respectively. (<b>c</b>) Histogram showing the number of serotonin immunoreactive cells in tamoxifen-treated <i>Tph2</i>^{<i>flox/null</i>}::<i>CMV-CreER</i>^<i>T</i> mice, as compared to wild-type and vehicle-treated <i>Tph2</i>^{<i>flox/null</i>}::<i>CMV-CreER</i>^<i>T</i> control mice. On the right, three representative coronal sections of B8-B9 and B7 <i>raphe</i> nuclei showing the distinct levels where serotonin positive neurons were counted. While the number of serotonin immunoreactive cells is unchanged between vehicle-treated <i>Tph2</i>^{<i>flox/null</i>}::<i>CMV-CreER</i>^<i>T</i> and wild-type animals, a progressive and rapid reduction of serotonin immunoreactive neurons is observed after tamoxifen treatment, resulting in brain serotonin depletion starting from 7 days after the end of the treatment. Data are presented as mean ± SEM. Scale bar: 400 μm.</p

<i>Pet1₂₁₀-Cre</i> transgene specifically identify <i>Pet1</i>-expressing, non-serotonergic <i>raphe</i> neurons.

<p>Double ISH performed on serial coronal sections of adult <i>Pet1₂₁₀-Cre/ROSA26YFP</i> mouse brains at the level of B8 <i>raphe</i> nucleus using combination of <i>Pet1</i>/<i>Tph2</i> (<b>a-a’’</b>), <i>YFP</i>/<i>Tph2</i> (<b>b-b’’</b>), <i>YFP</i>/<i>Pet1</i> (<b>c-c’’</b>) and <i>Tph2</i>/<i>Pet1</i> (<b>d-d’’</b>) riboprobes. In each combination the former probe is highlighted using the NBT/BCIP substrate, while the latter using the Fast Red chromogen. Boxed areas are shown at higher magnification in brightfield (<b>a’</b>, <b>b’</b>, <b>c’</b>, <b>d’</b>) or fluorescence (<b>a’’</b>, <b>b’’</b>, <b>c’’</b>, <b>d’’</b>). While all <i>Pet1</i>-positive cells also express <i>YFP</i> (arrowheads in <b>c’-c’’</b>), the presence of a <i>Pet1</i>-positive, non-serotonergic cell population was confirmed with all the other probe combinations (arrows in <b>a’-a’’</b>, <b>b’-b’’</b>, <b>d’-d’’</b>). Scale bar: 100 µm (<b>a</b>–<b>d</b>), 25 µm (<b>a’</b>-<b>a’’</b>, <b>b’</b>-<b>b’’</b>, <b>c’</b>-<b>c’’d’</b>-<b>d’’</b>).</p

Two-Photon Polymerization of Sub-micrometric Patterned Surfaces: Investigation of Cell-Substrate Interactions and Improved Differentiation of Neuron-like Cells

Direct Laser Writing (DLW) is an innovative tool that allows the photofabrication of high resolution 3D structures, which can be successfully exploited for the study of the physical interactions between cells and substrates. In this work, we focused our attention on the topographical effects of submicrometric patterned surfaces fabricated <i>via</i> DLW on neuronal cell behavior. In particular, we designed, prepared, and characterized substrates based on aligned ridges for the promotion of axonal outgrowth and guidance. We demonstrated that both rat PC12 neuron-like cells and human SH-SY5Y derived neurons differentiate on parallel 2.5 μm spaced submicrometric ridges, being characterized by strongly aligned and significantly longer neurites with respect to those differentiated on flat control substrates, or on more spaced (5 and 10 μm) ridges. Furthermore, we detected an increased molecular differentiation toward neurons of the SH-SY5Y cells when grown on the submicrometric patterned substrates. Finally, we observed that the axons can exert forces able of bending the ridges, and we indirectly estimated the order of magnitude of these forces thanks to scanning probe techniques. Collectively, we showed as submicrometric structures fabricated by DLW can be used as a useful tool for the study of the axon mechanobiology

Phenotypic traits of mutants at birth: impaired oro-buccal behavior and increased tidal volume

<p><b>Copyright information:</b></p><p>Taken from "Distinct roles of and in the development of rhythmic neural networks controlling inspiratory depth, respiratory frequency, and jaw opening"</p><p>http://www.neuraldevelopment.com/content/2/1/19</p><p>Neural Development 2007;2():19-19.</p><p>Published online 26 Sep 2007</p><p>PMCID:PMC2098766.</p><p></p> Plethsymographic recordings of wild-type (top), and heterozygous (middle) and homozygous (bottom) mutant mice at P0. Inspiration is upward. Note that in mice, there is a two-fold increase in tidal volume compared with and wild-type littermates, whereas the frequency is the same (about 110 breaths/minute). Individual data relating tidal volume (V, abscissa) and number (nb) of jaw openings (ordinates) at P0.1. Each symbol corresponds to one animal. Black triangles are for mutants (b, c), open circles represent mutants (c) and open squares correspond to wild-type animals (b). Note that mutants can be separated from other genotypes at P0.1, due to their two-fold increased tidal volume and their reduced number of jaw openings. Broken lines indicate the values used to calculate penetrance of the phenotype (V, all data inferior to mean – 1 standard deviation; jaw openings, all data superior to mean + 1 standard deviation)

A <i>Tph2</i>^<i>GFP</i> Reporter Stem Cell Line To Model <i>in Vitro</i> and <i>in Vivo</i> Serotonergic Neuron Development and Function

Modeling biological systems <i>in vitro</i> has contributed to clarification of complex mechanisms in simplified and controlled experimental conditions. Mouse embryonic stem (mES) cells can be successfully differentiated toward specific neuronal cell fates, thus representing an attractive tool to dissect, <i>in vitro</i>, mechanisms that underlie complex neuronal features. In this study, we generated and characterized a reporter mES cell line, called <i>Tph2</i>^<i>GFP</i>, in which the vital reporter GFP replaces the <i>tryptophan hydroxylase 2</i> (<i>Tph2</i>) gene. <i>Tph2</i>^<i>GFP</i> mES cells selectively express GFP upon <i>in vitro</i> differentiation toward the serotonergic fate, they synthesize serotonin, possess excitable membranes, and show the typical morphological, morphometrical, and molecular features of <i>in vivo</i> serotonergic neurons. Thanks to the vital reporter GFP, we highlighted by time-lapse video microscopy several dynamic processes such as cell migration and axonal outgrowth in living cultures. Finally, we demonstrated that predifferentiated <i>Tph2</i>^<i>GFP</i> cells are able to terminally differentiate, integrate, and innervate the host brain when grafted <i>in vivo</i>. On the whole, the present study introduces the <i>Tph2</i>^<i>GFP</i> mES cell line as a useful tool allowing accurate developmental and dynamic studies and representing a reliable platform for the study of serotonergic neurons in health and disease