Figure 1.
NAA is significantly reduced in NET KO mice.
Summary of results from MRS showing metabolite concentrations in the striatum relative to creatine plus phosphocreatine are shown for both cohorts of mice. N-acetyl aspartate (NAA) is significantly different (P<0.05; Student's t-Test for independent means; n = 11–12) between genotypes, with NAA for NET KO mice being 36% less than the wildtype mice.
Figure 2.
NET KO mice display a disorganized Striatal-GP interface.
Representative sections from a wildtype littermate (A and C) and a NET KO mouse that were embedded in register in the same block, sectioned and stained for thionine-Nissl (nuclei; A and B) and Solochrome (C and D). Three animals from each genotype were included in the same block, sectioned in register and stained in the same cup to ensure consistency and accuracy. This co-embedding, co-sectioning procedure yields the highest degree of precision correlations currently available for co-registration of histologic sections, which is of necessity less precise than MR dataset computational alignment. Despite this caveat, a distinct difference in axonal organization and bundling is observed in all 6 brains examined. In (A–B) Arrows indicate the striatum/globus pallidus interface. Note the size and regularity of the axonal swirls in (A) and the lack of regularity in (B). In (C–D) the blue-stained axonal bundles in the striatum of wildtype (arrow heads) are of uniform size and shape while in the KO (D) they are irregularly sized bundles. Also note the presence of scattered bundles in the globus pallidus (GP, outlined with dashed lines) in KO mouse (D) that are not seen in WT littermate (C). The lateral boundary of the internal capsule (IC) is less distinct in the KO mouse as well. While in these representative images some of these differences between WT and KO mice are accentuated by a slightly different angle of the histologic section, such alterations in KO mice were obvious in all three specimens regardless of slight differences in the sectioning plane.
Figure 3.
Tensor based morphometry indicates small local volume changes between NET KO mice and wildtype littermates.
FDR corrected at p<0.005 statistical parametric maps identifying local fractional volume changes necessary to transform NET KO to wildtype; red denotes regions where NET KO is contracted relative to wildtype and blue denotes regions where NET KO is expanded relative to wildtype. Color bar to the left indicates local fractional volume changes (e.g. 1 implies no change, 0.5 indicates 50% reduction, 2 indicates factor of 2 expansion). Arrows on the sagittal slice indicate locations of horizontal and axial slices. The thalmus (Th) and internal capsule (IC) are indicated. Other specific anatomical regions can be identified using Hoff et al. [109], the Allen Brain Atlas [110] (http://www.brain-map.org/welcome.do), or The Mouse Brain Library (http://www.mbl.org/atlas/atlas.php). Scale bar = 1 mm.
Figure 4.
Injection site locations are similar in wildtype and NET KO mice.
Injection site locations, represented as a sphere for each animal of the 23 mice used in the statistical analysis of prefrontal cortex injections, confirms accurate placement for both groups (blue: wildtype littermates, red: NET KO). Injection site locations have been overlaid onto axial, transverse and sagittal semi-transparent rendered images of the MRI template (Scale bar = 1 mm).
Figure 5.
Histology of the injection site showing precise location and minimal damage.
Left panel: A representative example of one brain section through the forebrain stained with Solochrome for both nuclei and fiber tracks and imaged at low magnification shows the injection site in the right frontal cortex (boxed region). Although the co-injected oil droplet identifying the injection site, leaves a small displacement, there is no bleeding, gliosis, or other indication of injury. Scale bar = 200 μm. Right panel: Fluorescent micrograph of an adjacent section of the same brain. The injection site is detected by fluorescent signal from the co-injected rhodamine-dextran tracer (red). Note that the tracer is confined to the region of the oil droplet and a few microns above it. Some auto-fluorescence of red blood cells within parenchymal capillaries is scattered in the background. Anatomical features revealed by a counter-stain for cellular nuclei with DAPI (blue) confirm that the location is within the forebrain. Scale bar = 20 μm.
Figure 6.
Statistical parametric maps show the progression of Mn2+ accumulation over time.
Representative sections through the statistical parametric maps show the progression of Mn2+ accumulation over time. Gray background is pre-injection MDT, while the green (1 hr > pre-injection), red (4 hr >1 hr), yellow (8 hr >4 hr) and blue (26 hr >8 hr) overlays indicate areas with increased intensity (FDR corrected at p<0.001) compared to the preceding time point. Representative axial (A), sagittal (B), and transverse (C) sections are displayed. Videos in Supplemental Information show the complete image data sets in each orientation for each genotype and in 3D (S3, S4, S5). Slice locations are indicated relative to Bregma for axial, relative to midline for sagittal, and relative to the brain surface for transverse sections are indicated to the left in each panel. Specific anatomical regions can be identified using Hoff et al. [109], the Allen Brain Atlas [110] (http://www.brain-map.org/welcome.do), or The Mouse Brain Library (http://www.mbl.org/atlas/atlas.php). Scale bars = 1 mm.
Table 1.
Comparison of anatomical structures highlighted by Mn2+ over time in NET KO and wildtype littermate mice; and at one day post Mn2+ injection in DAT and SERT KO mice and their respective wildtype controls.
Figure 7.
Schematic representation shows PFC associated circuits are somewhat more robust in WT compared to NET KO mice.
Major circuits delineated by MEMRI are shown for NET, DAT, and SERT KO mice compared with wildtype littermates. Red (WT) and blue (DAT KO) arrows in the schematic diagram indicate Mn2+ transport and accumulation in the different genotypes. Detailed anatomy and timing of Mn2+ induced image changes are shown in Table 1.