Subicular hypotrophy in fetuses with Down syndrome and in the Ts65Dn model of Down syndrome

Abstract

Intellectual disability in Down syndrome (DS) has been attributed to neurogenesis impairment during fetal brain development. Consistently with explicit memory alterations observed in children with DS, fetuses with DS exhibit neurogenesis impairment in the hippocampus, a key region involved in memory formation and consolidation. Recent evidence suggests that the subiculum plays a unique role in memory retrieval, a process that is also altered in DS. While much attention has been devoted to the hippocampus, there is a striking lack of information regarding the subiculum of individuals with DS and DS models. In order to fill this gap, in the current study, we examined the subiculum of fetuses with DS and of the Ts65Dn mouse model of DS. We found that in fetuses with DS (gestational week: 17-21), the subiculum had a reduced thickness, a reduced cell density, a reduced density of progenitor cells in the ventricular zone, a reduced percentage of neurons, and an increased percentage of astrocytes and of cells immunopositive for calretinin-a protein expressed by inhibitory interneurons. Similarly to fetuses with DS, the subiculum of neonate Ts65Dn mice was reduced in size, had a reduced number of neurons and a reduced number of proliferating cells. Results suggest that the developmental defects in the subiculum of fetuses with DS may underlie impairment in recall memory and possibly other functions played by the subiculum. The finding that the subiculum of the Ts65Dn mouse exhibits neuroanatomical defects resembling those seen in fetuses with DS further validates the use of this model for preclinical studies.

Keywords: Down syndrome; GFAP; NeuN; Ts65Dn mouse; calretinin; fetuses; neurogenesis; subiculum.

Figure 1

Anatomy of the subiculum in euploid fetuses and fetuses with DS. A–E. Representative images of Nissl‐stained coronal sections across the hippocampal region of a euploid fetus (A, C, E; case 164) and a fetus with DS (B, D; case 166). Images in (C) and (D) are higher magnification of the boxed areas in (A) and (B), respectively. The image in (E) is a higher magnification of the boxed region in (C) and shows the layers of the subiculum, the ventricular zone, and the intermediate zone. The black arrowhead in (A–D) indicates the border between the subiculum and presubiculum. The region between the black arrowhead and the white arrowhead in (C) and (D) corresponds to the part of the subiculum examined in this study. The double‐headed arrows in (C) and (D) indicate the location where the thickness of the molecular layer (black arrow) and pyramidal layer (white arrow) were measured. Calibration bar: A, B = 1000 µM; C, D = 500 µM; E = 200 µM. F–H. Thickness (F) and cell density (G) of the pyramidal layer, and thickness (H) of the molecular layer of the subiculum of fetuses with DS (n = 6) and euploid fetuses (n = 6). Values are mean ± SD. *P < 0.05; ***P < 0.001 (two tailed t‐test). Abbreviations: CA1 and CA3 = hippocampal fields; CS = collateral sulcus; d = dorsal; DG = dentate gyrus; DS = Down syndrome; ENT = entorhinal cortex; EU = euploid; IZ = intermediate zone; l = lateral; LV = lateral ventricle; m = medial; Mol = molecular layer; Pyr = pyramidal layer; PRES = presubiculum; SUB = subiculum; v = ventral; VZ = ventricular zone.

Figure 2

Density of proliferating cells in the subiculum and subicular VZ in euploid fetuses and fetuses with DS. A. Coronal section across the hippocampal region of a euploid fetus (case 104) immunostained for Ki‐67 and counterstained with Mayer’s haematoxylin. B, C. Higher magnification images of the regions enclosed by squares in (A), rotated approximately 45° counterclockwise. Cells immunostained for Ki‐67 appear labeled in brown: see, for instance, the cells indicated by the red arrowheads. D, E. Examples of sections immunostained for Ki‐67 and counterstained with Mayer’s haematoxylin from the ventricular zone overlying the subiculum in a euploid fetus (D) and a fetus with DS (E). Images are from cases 104 (D) and 203 (E). The white star indicates the border of the VZ that faces the lateral ventricle. Calibration bar: A = 500 µM; B, C = 50 µM; D, E = 25 µM. F, G. Density of Ki‐67‐positive cells, expressed as number of cells/mm² in the ventricular zone (F) and pyramidal layer plus intermediate zone (G) of the subiculum of fetuses with DS (n = 5) and euploid fetuses (n = 5). Values are mean ± SD. (*)P < 0.06; *P < 0.05 (two tailed t‐test). Abbreviations: CA1 and CA3 = hippocampal fields; d = dorsal; DG = dentate gyrus; DS = Down syndrome; EU = euploid; IZ = intermediate zone, l = lateral; LV = lateral ventricle; m = medial; PRES = presubiculum; Pyr = pyramidal layer; SUB = subiculum; v = ventral; VZ = ventricular zone.

Figure 3

Percentage of neurons and astrocytes in the subiculum of euploid fetuses and fetuses with DS. A. Sections immunostained for NeuN and counterstained with hematoxylin from the subiculum of a euploid fetus (case 27) and a fetus with DS (case 46). The red arrowhead and the black arrowhead mark a NeuN‐positive and a NeuN‐negative cell, respectively. B. Sections immunostained for GFAP and counterstained with hematoxylin from the subiculum of a euploid fetus (case 178) and a fetus with DS (case 133). The yellow arrowhead and the black arrowhead mark a GFAP‐positive and a GFAP‐negative cell, respectively. Calibration bar: A, B = 20 µM. C–E. Percentage of NeuN‐positive cells (C), GFAP‐positive cells (D) and cells that did not express either NeuN or GFAP (E) over total cell number in the pyramidal layer of the subiculum of fetuses with DS (n = 4) and euploid fetuses (n = 3). Values are mean ± SD. **P < 0.01 (two tailed t‐test). Abbreviations: DS = Down syndrome; EU = euploid.

Figure 4

Density of calretinin‐positive cells in the subiculum and subicular ventricular zone in euploid fetuses and fetuses with DS. A–D. Examples of calretinin‐positive cells in the ventricular zone (A) and pyramidal layer (C) of the subiculum of a euploid fetus (case 118), and in the ventricular zone (B), and pyramidal layer (D) of the subiculum of a fetus with DS (case 203). Sections were immunostained for calretinin and counterstained with hematoxylin. Colors have been inverted in order to better show calretinin‐positive cells (blue cells indicated by red arrowheads). The white star in (A) and (B) indicates the border of the ventricular zone facing the ventricle. Calibration bar: A, B = 50 µM; C, D = 20 µM. E. Density of calretinin‐positive cells in the ventricular zone of the subiculum. F–H. Density of calretinin‐positive cells (F), calretinin‐negative cells (G) and ratio of calretinin‐positive over calretinin‐negative cells (H) in the subiculum of fetuses with DS (n = 3) and euploid fetuses (n = 3). Values are mean ± SD. (*)P < 0.06; *P < 0.05 (two tailed t‐test). Abbreviations: CR = calretinin; DS = Down syndrome; EU = euploid.

Figure 5

Anatomy of the subiculum in neonate mice. A–D. Nissl‐stained coronal sections across the subiculum of a euploid mouse aged 2 days. Sections in (A–D) show the subiculum at progressively more caudal levels going from (A) to (D). The dotted lines indicate the border of the subiculum with field CA1 and cortical areas. E, F. Higher magnification images showing the layers (E) and cytoarchitecture (F) of the subiculum. The dashed line in (E) encloses the pyramidal layer of the subiculum. Calibration bar: A–D = 500 µM; E = 100 µM; F = 50 µM. Abbreviations: CA1 and CA3 = hippocampal fields; d = dorsal; DG = dentate gyrus; l = lateral; LV = lateral ventricle; m = medial; Mol = molecular layer; PRES = presubiculum; Pyr = pyramidal layer; RSC = retrosplenial cortex; SUB = subiculum; TH = thalamus; v = ventral; WM = white matter.

Figure 6

Cellularity and proliferation rate in the subiculum of Ts65Dn and euploid mice. A–B. Nissl‐stained coronal sections across the subiculum of a euploid (A) and a Ts65Dn (B) mouse aged two days. Images, which were taken at approximately the same rostro‐caudal plane, show that the size of the subiculum is patently reduced in Ts65Dn mice. The dotted lines indicate the borders of the subiculum with field CA1 and the cortex. Calibration bar: A, B = 200 µM. C. Volume of the pyramidal layer of the subiculum (panel on the left), cell density (middle panel) and total number of cells (panel on the right) in the pyramidal layer of the subiculum of euploid (n = 5) and Ts65Dn (n = 5) mice. Values (mean ± SD) refer to one hemisphere. D, E. Sections immunostained for BrdU and counterstained with hematoxylin across the subiculum of a euploid (D) and a Ts65Dn (E) mouse. The dotted lines indicate the borders of the subiculum. Higher magnification images of the boxed areas are reported on the right. The red arrowhead shows a BrdU‐positive cell. Calibration bar = 100 µM (higher magnification images); 50 µM (lower magnification images). F, G. Total number of BrdU‐positive cells (upper panel) and mean number of BrdU‐positive cells per section (lower panel) in the pyramidal layer plus molecular layer of the subiculum in euploid (n = 5) and Ts65Dn (n = 5) mice. Values (mean ± SD) refer to on hemisphere. *P < 0.05; **P < 0.01 (two tailed t‐test). Abbreviations: CA1, CA3 = hippocampal fields; DG = dentate gyrus; Eu = euploid; LV = lateral ventricle; Pyr = pyramidal layer; Mol = molecular layer; SUB = subiculum; SVZ = subventricular zone.