Dynamic changes in the pattern of labeled cells mirror the neurogenic cascade in the DG.

<p>Immunohistological detection of eGFP⁺ cells in the DG of Nestin-Cre mice at indicated time points after injection of the LV-Cre-Flex vector. At early time points, mainly cells in the SGZ are labeled (upper panel). Arrowheads indicate cells with a typical RGC morphology. At later time points, also cells in the GCL are labeled (middle panel). From 3 months p.i. onwards, labeled mossy fibers projecting towards the CA3 can be detected (lower panel). Scale bar = 100 μm.</p

Long-term BLI of Nestin-Cre and Nestin-CreER^T2 mice after injection of the Cre-Flex LV vector in the DG.

<p>(<b>A</b>) Nestin-Cre mice (n = 9) injected with the Cre-Flex LV vector were imaged with BLI on a regular basis until 9 months p.i. The BLI time course was initially characterized by fluctuations and high data variability, followed by stabilization of the signal after 45 days p.i. (<b>B</b>) Representative BLI scans of a Nestin-Cre mouse followed over time. (<b>C,D</b>) Four days after stereotactic injection, eGFP⁺ cells were not only detected in the DG, but also in the CC and injection tract where they displayed a typical stellar morphology as shown (<b>D</b>). (<b>E</b>) Triple immunofluorescent staining for eGFP, GFAP and S100β showed labeling of reactive astrocytes in the CC and injection tract. (<b>F</b>) Cre recombination was induced in Nestin-CreER^T2 mice 3 weeks after injection of the Cre-Flex LV vector (n = 5), after which they were imaged regularly until 3 months post tamoxifen induction. The BLI kinetics of Nestin-CreER^T2 were comparable to those of Nestin-Cre mice. Data of Nestin-Cre mice are the same as in (<b>A</b>). Scale bars: (<b>C)</b> = 250 μm; (<b>D,E</b>) = 50 μm.</p

Phenotypical analysis of different labeled cell types in the DG.

<p>Upper and middle panel: Triple fluorescent stainings for eGFP/Sox2/GFAP, eGFP/DCX/NeuN and eGFP/GFAP/S100β followed by confocal analysis allowed to identify different labeled cell types as indicated. Lower panel: time-dependent decrease of DCX expression in the DG. Scale bars = 50 μm.</p

Long-term fate-mapping analysis in the DG of Nestin-Cre mice.

<p>(<b>A</b>) The proportion of different labeled cell types was determined at different time points p.i. The number of eGFP⁺ cells that was analyzed for co-localization with different phenotypical markers was 146±36 at 10 days p.i. (n = 4), 265±28 at 1 month p.i. (n = 3), 454±129 at 3 months p.i. (n = 3) and 647±48 at 9 months p.i. (n = 3). The proportion of eGFP⁺ RGCs (<i>p</i> = 0.01) and neuroblasts (<i>p</i> = 0.02) decreased over time, while the proportion of eGFP⁺ mature neurons increased (<i>p</i> = 0.01). Raw data are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143772#pone.0143772.s002" target="_blank">S2 Table</a>. (<b>B</b>) The absolute number of labeled cells within each population was calculated by multiplying the population ratio per animal by the absolute number of the eGFP⁺ cells as determined by stereology in the same animal. The number of eGFP⁺ RGCs initially decreased (<i>p</i> = 0.03) but remained constant after 1 month p.i. The number of eGFP⁺ neuroblasts decreased from 10 days to 9 months p.i. (<i>p</i> = 0.04). The number of eGFP⁺ mature neurons continuously increased over time (<i>p</i> = 0.01). Raw data are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143772#pone.0143772.s003" target="_blank">S3 Table</a>.</p

A time-dependent increase in number of eGFP-labeled cells is not paralleled by a significant increase in BLI signal.

<p>(<b>A</b>) Stereological quantifications of the number of eGFP⁺ cells in the DG of Nestin-Cre mice perfused at the indicated time points after injection of the LV-Cre-Flex vector. The number of eGFP⁺ cells continuously increased over time (<i>p</i> < 0.0001). At 9 months p.i., the number of eGFP⁺ cells was significantly higher compared to 4 days p.i. (p < 0.001), to 10 days p.i. (<i>p</i> < 0.001), to 1 month p.i. (<i>p</i> < 0.01) and to 3 months p.i. (<i>p</i> < 0.05). Raw data are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143772#pone.0143772.s001" target="_blank">S1 Table</a>. (<b>B</b>) Histological detection of eGFP⁺ cells at indicated time points. Scale bar = 250 μm. (<b>C</b>) Quantification of the BLI signal from the same animals depicted in (<b>A</b>). There was a trend towards a time-dependent increase of BLI signal but this was not significant (<i>p</i> = 0.25). (<b>D</b>) The number of eGFP⁺ cells per animal was correlated to the BLI signal of the same animal. A significant (Pearson correlation <i>p</i> = 0.007, n = 21) but weak (R² = 0.32) correlation was evident.</p

Cre-Flex LV vectors label a restricted cell population in the DG of Nestin-Cre mice.

<p>(<b>A-C</b>) WT mice were injected with a constitutive LV-eGFP-T2A-Fluc vector in the DG (n = 3). (<b>A</b>) Representative BLI image 6 days p.i. (<b>B-D</b>) Histological detection of eGFP⁺ cells across the DG including the hilus, the SGZ and the GCL (<b>C-D</b>). Labeled mature neurons in the GCL extend dendrites into the molecular layer and axons into the hilus that eventually project towards the CA3 (<b>B)</b>. (<b>D</b>) Schematic overview of the different regions of the DG. (<b>E-H</b>) Transgenic Nestin-Cre mice were injected with a LV-Cre-Flex>E) Representative BLI image 10 days p.i. (<b>F-G</b>) Histological detection of eGFP⁺ cells that mainly reside in the SGZ. (<b>H</b>) Histological detection of Cre⁺ cells in the SGZ of Nestin-Cre mice. (<b>I-L</b>) WT mice were injected with a LV-Cre-Flex>I) Representative BLI image 14 days p.i. (<b>J-K</b>) Histological detection of eGFP⁺ cells reveals very sparse labeling in the DG. (<b>L</b>) Absence of Cre⁺ cells in the DG of WT mice. Scale bar (<b>B,F,J</b>) = 250 μm; (<b>C,G,H,K,L</b>) = 100 μm.</p

Schematic representation of the functional PET atlases construction pathway.

<p>The full arrows express the followed procedure, the arrows in dotted line the objective. On the standardized atlases a stereotactic grid is shown (2 mm interlines).</p

Construction and Evaluation of Quantitative Small-Animal PET Probabilistic Atlases for [¹⁸F]FDG and [¹⁸F]FECT Functional Mapping of the Mouse Brain

<div><p></p><p>Automated voxel-based or pre-defined volume-of-interest (VOI) analysis of small-animal PET data in mice is necessary for optimal information usage as the number of available resolution elements is limited. We have mapped metabolic ([¹⁸F]FDG) and dopamine transporter ([¹⁸F]FECT) small-animal PET data onto a 3D Magnetic Resonance Microscopy (MRM) mouse brain template and aligned them in space to the Paxinos co-ordinate system. In this way, ligand-specific templates for sensitive analysis and accurate anatomical localization were created. Next, using a pre-defined VOI approach, test-retest and intersubject variability of various quantification methods were evaluated. Also, the feasibility of mouse brain statistical parametric mapping (SPM) was explored for [¹⁸F]FDG and [¹⁸F]FECT imaging of 6-hydroxydopamine-lesioned (6-OHDA) mice.</p><p>Methods</p><p>Twenty-three adult C57BL6 mice were scanned with [¹⁸F]FDG and [¹⁸F]FECT. Registrations and affine spatial normalizations were performed using SPM8. [¹⁸F]FDG data were quantified using (1) an image-derived-input function obtained from the liver (cMR_glc), using (2) standardized uptake values (SUV_glc) corrected for blood glucose levels and by (3) normalizing counts to the whole-brain uptake. Parametric [¹⁸F]FECT binding images were constructed by reference to the cerebellum. Registration accuracy was determined using random simulated misalignments and vectorial mismatch determination.</p><p>Results</p><p>Registration accuracy was between 0.21–1.11 mm. Regional intersubject variabilities of cMR_glc ranged from 15.4% to 19.2%, while test-retest values were between 5.0% and 13.0%. For [¹⁸F]FECT uptake in the caudate-putamen, these values were 13.0% and 10.3%, respectively. Regional values of cMR_glc positively correlated to SUV_glc measured within the 45–60 min time frame (spearman r = 0.71). Next, SPM analysis of 6-OHDA-lesioned mice showed hypometabolism in the bilateral caudate-putamen and cerebellum, and an unilateral striatal decrease in DAT availability.</p><p>Conclusion</p><p>MRM-based small-animal PET templates facilitate accurate assessment and spatial localization of mouse brain function using VOI or voxel-based analysis. Regional intersubject- and test-retest variations indicate that for these targets accuracy comparable to humans can be achieved.</p></div

Statistical parametric maps of 6-OHDA mice.

<p>Differences for the brain-regions have been color-coded and are superimposed on the MRM template. Series of axial sections with t-maps rendered on the MRM atlas of the mouse brain show significant reductions in glucose metabolism (a) and DAT availability (b). The colored bars on the right express T-score levels. The intersection points of the axial planes have been set to the position of the right caudate-putamen, i.e. (x,y) = (−1.8, 0.2) and (x,y) = (−2.0, 0.0) for [¹⁸F]FDG and [¹⁸F]FECT, respectively. Images are in radiological convention.</p

Estimates of registration accuracy for a simulated [¹⁸F]FDG and [¹⁸F]FECT image of a control and a 6-OHDA-lesioned animal, derived from the VOI map, after re-registration of randomly misaligned data.

<p>All values are expressed in mm.</p