The Ts65Dn genotype was confirmed by PCR and the phenotype by histologic examination of individual mice fixed by perfusion after MRI (). Ts65Dn mice displayed the expected morphological phenotype of decreased cell numbers and loss of cholinergic neurons in the septal nuclei (), as previously reported (Granholm et al., 2000
, Cooper et al., 2001
, Seo and Isacson, 2005
). Histological examination revealed qualitative decreases in the number and size of cells in the septal nuclei of DS mice as compared to 2N littermates (). Immunohistochemistry with anti-choline acetyl transferase (ChAT) demonstrated diminished numbers of cholinergic neurons in the medial septal nuclei (MSN) of DS mice versus 2N littermates (). In six of the mice, counts of ChAT positive nuclei in histologic sections spanning the MSN demonstrated a 30 ± 5% decrease in the number of neurons in DS versus 2N littermates. ChAT-positive neuronal cell bodies found in the MSN of DS brains also appeared shrunken compared to the plump cell bodies in littermates. No obvious differences in thickness or number of fibers in the hippocampal commissure, fimbria, or fornix were observed in histological sections. Thus, the aging Ts65Dn mice used in this study displayed the expected morphological abnormalities previously described for these mice, features similar to those found in human Alzheimer's disease and Down syndrome.
DS mice have fewer cholinergic neurons in the MSN
Injection site analysis by correlation of optical and MR microscopy
Co-injection of the tracer, RDA, with the Mn2+ allowed us to correlate the injection site in histologic sections with MR images of the same individuals. Imaging by combined phase and fluorescent microscopy of unstained gelatin-embedded sections of brains fixed immediately after acquisition of the last MR image (24 hr) identified the precise location of the injection (). In animals fixed 6 days after injection, no needle track or tissue damage at the injection sites were identified.
Injection site visualized in histologic sections showing lack of tissue damage
Comparison of MR images captured 0.5 hr after injection demonstrated that the injection position was accurately and reproducibly placed in all the mice studied (). The epicenter of the injection site (±200μm diameter), containing the highest concentration of Mn2+
, appears hypointense (dark) in MR images at this early time point because the level of Mn2+
there was sufficient to produce significant T2
-shortening and concomitant darkening (Lee, 1991
). Analysis of the 3D location of this epicenter in each of the 13 animals demonstrated that among all animals injections were routinely placed in CA3 of the hippocampus (see Materials and Methods).
At 0.5 hr post injection, the hypointense injection site was surrounded by a white halo of hyperintensity indicating passive diffusion of the Mn2+
ion outward from the injection site. The halo volume was found to be 9.3±1.9 μl for the Ts65Dn cohort and 9.0±1.4 μl for the 2N littermates. An equivalent spherical volume would have a radius of 1.3 mm. Such diffusion is a common factor in all studies employing water-soluble low molecular weight tracers. Previous studies in the songbird, mouse, rat, minipig, and macaque note the same initial Mn2+
diffusion with no lack of specificity of subsequent projection patterns (Saleem et al., 2002
, Van der Linden et al., 2002
, Leergaard et al., 2003
, Pautler et al., 2003
, Van der Linden et al., 2004
, Watanabe et al., 2004b
, Jelsing et al., 2006
, Murayama et al., 2006
). Presumably, only neurons in the immediate vicinity of the high Mn2+
concentration take up enough Mn2+
for detection after transport to distant sites. To focus the high concentration of Mn2+
in as small a brain region as possible, the volume of Mn2+
injected in this study (4.3 nl) was 10-1000 times less than that employed in previous studies. The reproducible placement of the injection sites and the comparable size of the diffusion halo argue that consistent amounts of both tracers, Mn2+
and RDA, were delivered to the same population of neurons in each animal tested. Thus, comparisons between individual animals or across cohorts are not confounded by effects due to different amounts of tracer introduced or different locations being probed.
Histological tract tracing demonstrated that the injection site was appropriate for uptake into the hippocampal-forebrain circuit
Co-injection of RDA with the Mn2+
allowed tracing of fluorescence in histologic sections of the same animals imaged by MR (). By comparing the patterns of distribution of Mn2+
and RDA, we confirmed that the placement of the injection site was appropriate for entry of tracer into the hippocampal-septal connections, and that the transport within those connections was operational. Small RDA such as the 3kD used here are taken up and moved inside neuronal processes, either diffusively or primarily in the retrograde direction at slow rates. Diffusive motion of small dextrans is observed in the squid axon (Terasaki et al., 1995
) and 1-3 weeks is required for studies of CNS pathways in adult rodents (Reiner et al., 2000
, Van Haeften and Wouterlood, 2000
). In serial sections of brains fixed 6 days after injection, RDA was found along the fimbria and fornix ipsilateral to the injection site but not detectable on the contralateral, non-injected, side (). At the level of the medial septal nucleus, containing the cholinergic neurons that send processes to the hippocampus, RDA fluorescence appeared on both sides of the midline ().
Transport of 3kD rhodamine-dextran-amine, a traditional tract tracer, from hippocampus to medial septum after co-injection with the Mn2+ into the right hippocampus
These results confirm that the injection site in the hippocampus was positioned appropriately for entry into the hippocampal-septal pathway. No other locus with detectible accumulation of RDA was found. These results did not reveal any obvious difference in RDA fluorescence between DS mice and their 2N littermates in the fimbria, fornix or septal region. This is consistent with reports of another retrograde tracer, Fluorogold, which was injected in CA3 of the hippocampus in much higher quantities than in our study, and arrived into the MSN of Ts65Dn and their littermates in similar amounts (Salehi et al., 2006
Mn2+ enhanced signal appears in the septal region within 6 hours of injection
Qualitative assessment of MR slices taken at the level of the basal forebrain revealed a time-dependent accumulation of Mn2+ in the septal nuclei after injection into the hippocampus (). These coronal slices were selected from 3D MR images at Bregma +0.4 mm position (see for diagram). In these averaged 3D images at 0.5 hr post-injection, no Mn2+ enhanced brightness was seen in the septal region although slices through the injection site at Bregma -4 .0 mm display Mn2+ enhancement as shown in . In contrast, by 6 hr the septal region ipsilateral to the injection site appeared brighter and this persisted in the 24 hr image (). Mn2+ enhancement in the septal region at 6 and 24 hr is qualitatively similar or greater in Ts65n mice than in 2N littermates. Mn2+ enhanced signal also appeared in the contralateral hippocampus at 24 hr as shown in coronal slices at the level of the dentate gyrus of the hippocampus, Bregma -2.7 (). Again this enhancement was qualitatively similar or greater in Ts65Dn than in 2N littermates.
was transported from the injection site in the hippocampus to the septum in relatively short times and to the contralateral hippocampus within 24 hours. Transport from the injection site to the septum required more than 0.5 hr and less than 6 hr to traverse the distance of ~6.5 mm (from injection site, along the fimbria, to septum); we thus calculate a transport rate from 0.3 to 3.6 μm/sec, consistent with fast axonal transport of 0.05-2 μm/sec (Satpute-Krishnan et al., 2006
Mn2+ is transported further faster in Ts65Dn mice than in normal littermates
SPM along with spatial realignment and normalization have become standard methodologies for human brain imaging studies (Thompson and Toga, 1996
, Thompson et al., 1997
, Woods et al., 1998
, Hajnal et al., 2001
, Toga and Thompson, 2001
, Toga and Mazziotta, 2002
, Lancaster et al., 2003
, Yoo, 2004
). The same general procedures employed here have been used to make ‘standard atlas’ spaces for mouse brains of various strains and ages (MacKenzie-Graham et al., 2004
, Kovacevic et al., 2005
, Lee et al., 2005
). Despite significant anatomical abnormalities (Baxter et al., 2000; Kurt et al., 2000; Olson et al., 2004
), the brains of the Ts65Dn mice were surprisingly similar structurally, and could be aligned into the same 3D space. However, these Ts65Dn abnormalities precluded alignment of the mutant brains into the same template as their anatomically normal littermate controls. We therefore pursued analysis of the circuits enhanced by Mn2+
within each cohort and compared the resulting patterns between groups.
To identify dynamic changes in the anatomical pattern of Mn2+ enhancement over time after injection, we used SPM to compare each subsequent dataset with that immediately preceding it in the time series. Here we present three comparisons for each genotype: 0.5 hr post-injection versus pre-injection (), 6 hr versus 0.5 hr (), and 24 versus 6 hr post-injection (). Those voxels with significantly (P<0.0005) increased signal intensity in the later time point image are displayed as a colored voxel on the maps ().
Increased Mn2+ transport in the hippocampal-forebrain pathway is apparent in statistical parametric mapping analysis of the MR data
In the 0.5 hr versus pre-injection comparisons (), both sets of animals displayed similar paired Student's t-test maps at P<0.0005. The injection site was in a similar position and of a similar size in the posterior hippocampus in both cohorts. This result confirms the precise position of the injection site and that the amount of Mn2+ injected was similar in all animals. Relative lack of enhanced signal (colored voxels in ) outside the injection site attests to the dependence of signal enhancement (at P<0.0005) on the presence of Mn2+ and to the accuracy of the alignment.
Comparison of 6 hr versus 0.5 hr statistical maps demonstrated that Mn2+ followed the expected route from the hippocampus through the fimbria to the basal forebrain in both DS and 2N littermates (). Voxels with statistically significant increased intensity were seen in both normal and Ts65Dn cohorts in CA3, the fimbria (fi), the septofimbrial nucleus (Sfi), and the LSN (). In the DS cohort more statistically significant voxels were found in these structures and significant signal had progressed further along the expected pathways, appearing already at 6 hours in the MSN, contralateral fimbria, as well as secondary olfactory structures (dorsal portion of acumbens core, anterior olfactory nucleus, and dorsal tenia tecta; see ).
Table 1 Extent of Manganese Transport from Hippocampal Injection SiteMn2+ transports along expected pathways more rapidly in DS than in 2N littermates. Structures with statistically significant (p<0.0005) increase in MRI intensity when compared with the (more ...)
Continued transport in these living mice during the 6 hr to 24 hr interval was revealed by SPM comparisons of these two time points. In this case, only voxels whose intensity increased between 6 hr and 24 hr are displayed (). This comparison revealed that, by 24 hr in 2N littermates, Mn2+ transport caught up to that seen at earlier time points in DS mice. Thus by 24 hr Mn2+ had progressed into the MSN in both sets of animals. By this time, both cohorts also displayed voxels with significantly increased intensity in CA3 of the contralateral hippocampus, and bed of nucleus stria terminalis ().
In addition, at 24 hr post-injection, the DS group again out-performed their 2N littermates, displaying Mn2+ signal further rostrally along the hippocampal-forebrain pathway, with statistically significant signal enhancement in the nucleus accumbens and areas within the ventral pallidum; the ventromedial hypothalamus and the amygdala; and the anterior part of retrosplenal granular cortex ( and ). Thus DS mice compared to 2N littermates displayed voxels with statistically significant Mn2+ enhancement further along the hippocampal-forebrain pathway at both 6 and 24 hr after injection.
Mn2+ enhanced signal occupies more volume along the hippocampal-forebrain tract in DS mice than in littermates
Comparison of the total volume occupied by significantly enhanced voxels (P<0.0005) in the statistical parametric maps of each set of animals demonstrated that DS had a larger volume enhanced by Mn2+ than their 2N littermates (). In the map comparing the 6 hr to 0.5 hr data (), the volume of Mn2+ enhanced voxels in the basal forebrain occupied 2.4 mm3 in DS animals, but only 1.3 mm3 in 2N littermates. Mn2+ enhanced volumes at 24 hr in the contralateral hippocampus were 3.1 mm3 and 0.2 mm3 in the DS and 2N littermates, respectively. Thus, the extent of Mn2+ transport is greater (observed further along expected tracts) and more robust (more volume affected) in DS mice as compared to 2N littermates.
Volume occupied by statistically enhanced voxels is greater in DS than in normal brains