In vivo MRI identifies cholinergic circuitry deficits in a Down syndrome model

Abstract

In vivo quantitative magnetic resonance imaging (MRI) was employed to detect brain pathology and map its distribution within control, disomic mice (2N) and in Ts65Dn and Ts1Cje trisomy mice with features of human Down syndrome (DS). In Ts65Dn, but not Ts1Cje mice, transverse proton spin-spin (T(2)) relaxation time was selectively reduced in the medial septal nucleus (MSN) and in brain regions that receive cholinergic innervation from the MSN, including the hippocampus, cingulate cortex, and retrosplenial cortex. Basal forebrain cholinergic neurons (BFCNs) in the MSN, identified by choline acetyltransferase (ChAT) and nerve growth factor receptors p75(NTR) and TrkA immunolabeling were reduced in Ts65Dn brains and in situ acetylcholinesterase (AChE) activity was depleted distally along projecting cholinergic fibers, and selectively on pre- and postsynaptic profiles in these target areas. T(2) effects were negligible in Ts1Cje mice that are diploid for App and lack BFCN neuropathology, consistent with the suspected relationship of this pathology to increased App dosage. These results establish the utility of quantitative MRI in vivo for identifying Alzheimer's disease-relevant cholinergic changes in animal models of DS and characterizing the selective vulnerability of cholinergic neuron subpopulations.

Figure 1

Mean T₂ assessments in Ts65Dn, Ts1Cje, and 2N mice. Regional comparisons of mean T₂ values and MRI volumetric findings between 2N mice and Ts65Dn, Ts1Cje trisomic mice. Anatomical guidelines for outlining regions of interest (ROIs) were determined by comparing anatomical structures in the MRI slices with standard atlas {A: ROIs of cortex (a), corpus callosum (b), medial septal nucleus (c), striatum (d), and hippocampus (e)} (A). Detailed comparisons are available in bar graph B, indicating significant overall regional decrease in mean T₂ values ± SEM in Ts65Dn mice in the whole brain (WB; p < 0.05). Region-specific reduction in T₂ was identified in the hippocampus (HP; p < 0.05), medial septal nucleus (MS; p < 0.01), and the cingulate cortex (C(C); p< 0.05), but not in the corpus callosum (CC), striatum (ST), or brainstem (BS) (B). By contrast, as illustrated in bar graph in panel C, there was no significant difference of T₂ relaxation times in the WB or any regions in Ts1Cje mice.

Figure 2

Spatial distribution of VBT2 in Ts65Dn mice. Occurrence of VBT2 clusters of reduced T₂ identified by voxel-wise analysis of T₂ maps from Ts65Dn mice compared with 2N mice (A) or Ts1Cje mice compared with 2N mice (B). The red pixels represent voxels of reduced VBT2 (p < 0.05). In Ts65Dn mice, VBT2s are distributed predominantly within the cingulate cortex (Cg), retrosplenial cortex (Rsp), hippocampal subfields (CA1, and CA3), dentate gyrus (DG), and medial septal nucleus (MS). In contrast to Ts65Dn mice, Ts1Cje mice (B) exhibited fewer and randomly distributed VBT2s within the same brain regions. Computer-generated 3-D image renderings of VBT2s in Ts65Dn (C) and Ts1Cje (D) mice illustrate that in Ts65Dn mice, the VBT2 clusters are located predominantly within the cingulate cortex and retrosplenial cortex, whereas VBT2 clusters were fewer or absent in similar regions from Ts1Cje mice.

Figure 3

Expression of cholinergic markers within the MSN of Ts65Dn, Ts1Cje, and 2N mice. Immunocytochemical analysis of BFCNs within the MSN of 2N (A, D, G) and Ts1Cje (B, E, H) and Ts65Dn (C, F, I) trisomic mice revealed a decrease in the numbers of immunoreactive neurons in the MSN of Ts65Dn mice compared with Ts1Cje and 2N controls. This reduction in cholinergic phenotype was evident using established cholinergic markers including ChAT (A-C, arrows), high-affinity NGF receptor TrkA (D-F, arrows), and low-affinity NGF receptor p75^NTR (G-I, arrows). In Ts65Dn mice, many BFCNs in the MSN exhibited atrophic changes such as shrunken perikarya (L and L inset arrows), condensed or swollen nuclei and the appearance of apoptotic bodies detected by light microscopy using Nissl stain and by electron microscopy (not shown). No significant differences in BFCN number or morphology were seen in the MSN of Ts1Cje compared with 2N controls (K and K inset, J and J inset). Scale bar A-I: 200 μm, J-L 20 μm.

Figure 4

Loss of AChE expression in the hippocampus and cingulate cortex in Ts65Dn trisomic mice. Enzyme histochemical analysis of in situ AChE activity in the dorsal hippocampus (AC), cingulate cortex (D-F), and temporal cortex (G-I) of 2N (A, D, G), Ts1Cje (B, E, H), and Ts65Dn (C, F, I) mice. In Ts65Dn mice compared with Ts1Cje and 2N littermate controls, a qualitative decrease in the number of AChE reactive cholinergic fibers was detected in CA3 of the hippocampus (C, arrowheads) and in the cingulate cortex (F, F′, and F″), particularly in laminae III and V. F′ and F″ are high magnification views demonstrating the loss of AChE reactivity in lamina III. This loss of fiber staining, which reflects loss of cholinergic terminals in the hippocampus and cingulate cortex, was not evident in cingulate lamina III and V from 2N (D, D′, and D″) and Ts1Cje (E, E′, and E″) mice and could be attributed to the decrease in cholinergic projection neurons in the MSN (see Figure 3) leading to the loss of innervation to these brain regions. No differences in the density of AChE reactive fibers were seen throughout the laminae of temporal cortex from 2N control mice (G, G′) and Ts1Cje (H, H″) and Ts65Dn (I, I′) trisomic mice brains. Higher magnification images of lamina III of the temporal cortex, which receives its main cholinergic input from more posterior areas of the cholinergic system, are shown in panels G″-I″. Densitometric analysis of AChE activity ± SEM in the hippocampus, and cingulate cortex (J) revealed a significant decrease in the AChE-reactive fibers in Ts65Dn mice compared to 2N and Ts1Cje mice. Asterisk denotes (p < 0.05).

Figure 5

Alterations in MAP2 expression within Ts65Dn mice. In Ts65Dn mice, MAP2 immunocytochemistry revealed a qualitative increase in the levels of MAP2 immunostaining in the apical dendrites of pyramidal cells in layer V of the cingulate cortex (C, arrows) and within the hippocampal CA fields (F, arrows) compared to the levels of MAP2 dendritic staining in the cingulate cortex and hippocampus from 2N control (A, D, arrows) and Ts1Cje trisomic mice (B, E, arrows). In Ts65Dn mice, MAP2-immunoreactive dendrites of large pyramidal neurons were shorter and thicker and less tapered compared with 2N and Ts1Cje mice. No significant changes in the magnitude of MAP2 immunolabeling or in dendrite morphology were detected in the temporal cortex of Ts65Dn mice compared to 2N and Ts1Cje mice (G-I). Morphometric analysis (J) of MAP2 immunoreactivity in brain sections from Ts65Dn, Ts1CJe and 2N mice confirmed the qualitative immunocytochemical observations. Quantitative densitometric analysis of MAP2 immunoreactive protein (± SEM) measured by Western blot analysis (K) revealed an increase in MAP2 levels in the cingulate cortex and hippocampus, but not in the temporal cortex in Ts65Dn mice compared to 2N and Ts1Cje mice. Single asterisk denotes (p < 0.05) and double asterisk indicates (p < 0.01). Scale bar: 20 μm.

Figure 6

Schematic of VBT2 changes in Ts65Dn mice. Decreases in T₂ correlate with cholinergic deficits observed in Ts65Dn mice. Green dots show the distribution of VBT2 and red dots represent areas with a dramatic loss of the cholinergic phenotype. Red arrows indicate MSN cholinergic pathways and blue arrows indicate additional cholinergic pathways. Cholinergic deficits in Ts65Dn mice are located predominantly within the MSN as well as the projection path to the cingulate cortex and hippocampus, where VBT2 hypointensities also were prominent Key: CG, cingulate cortex; HDB, horizontal limb of the diagonal band; Hippo, hippocampus; NB, nucleus basalis; RS, retrosplenial cortex; SI, substantia innominata, VDB, ventral limb of the diagonal band.