# Dendritic phenotype and proliferation potency in the hippocampal dentate gyrus of the Ts66Yah model of Down syndrome

Marco Emili, Fiorenza Stagni, Sandra Guidi, Carla Russo, Claire Chevalier, Arnaud Duchon, Yann Hérault, Renata Bartesaghi

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## To cite this version:

Marco Emili, Fiorenza Stagni, Sandra Guidi, Carla Russo, Claire Chevalier, et al., Dendritic phenotype and proliferation potency in the hippocampal dentate gyrus of the Ts66Yah model of Down syndrome. Neuroscience Letters, 2025, 850, pp. 138156. 10.1016/j.neulet.2025.138156. hal-05078930

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Research article

# Dendritic phenotype and proliferation potency in the hippocampal dentate gyrus of the Ts66Yah model of Down syndrome

Marco Emili<sup>a</sup>, Fiorenza Stagni<sup>a</sup>, Sandra Guidi<sup>b</sup>, Carla Russo<sup>a</sup>, Claire Chevalier<sup>c</sup>, Arnaud Duchon<sup>c</sup>, Yann Herault<sup>c,d,*</sup>, Renata Bartesaghi<sup>b,* ID</sup>

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## ARTICLE INFO

**Keywords:**

Down syndrome

Ts66Yah model

Hippocampus

Dentate gyrus

Dendritic development

Dendritic spines

Neurogenesis

## ABSTRACT

The Ts65Dn mouse is the most widely used model of Down syndrome (DS), although, in addition to the triplication of 90 genes homologous to Human Chromosome 21 (Hsa21) genes, it bears the triplication of 46 extra genes. To clarify the latter's impact, the Ts66Yah model has been created from the Ts65Dn mouse by exploiting CRISPR/Cas9 technology for extra gene deletion. It has been found that, similar to the Ts65Dn model, the Ts66Yah model exhibits impairment in hippocampus-dependent learning and memory and age-related hippocampal deterioration, with no increased activity. We examine here the dendritic development of the hippocampal granule neurons and the proliferation potency of granule cell precursors in Ts66Yah mice because these phenotypes are impaired in Ts65Dn mice and individuals with DS starting from early life stages and are thought to underpin cognitive impairment. In Ts66Yah mice aged 15 days and those aged three months, we found no reduction in dendritic arborization, dendritic spine density, proliferation potency, or total number of granule cells, suggesting that other mechanisms may underpin the behavioral impairment found in the Ts66Yah model in adulthood. Thus, the Ts66Yah model is unsuitable to study these neurodevelopmental alterations, although it may be useful to study other DS-related phenotypes.

## Significance statement

This study provides novel information regarding hippocampal neurogenesis and dendritogenesis in the Ts66Yah mouse, a recently created model of Down syndrome.

## 1. Introduction

Down syndrome (DS) is a genetic pathology due to the triplication of Hsa21. Among the 833 genes harbored on Human Chromosome 21 (Hsa21), 232 protein-coding genes (PCGs) have been identified [1]. The genetic imbalance due to Hsa21 triplication causes somatic alterations that may be inconsistently present [2]. Alteration of brain development and associated intellectual disability (ID), however, are consistent

phenotypic features of DS [3]. To shed light on these issues, numerous mouse models of this pathology have been created. As reported in many DS partial trisomy models [1], the overdosage of several genes has been shown to cause ID but potential therapeutic approaches to counteract ID are still to be found.

The Ts65Dn mouse [Ts(17<sup>16</sup>)65Dn] is one of the first and most used DS models [4], because its phenotypes recapitulate several features of DS. This model bears triplication of 90 PCGs harbored on mouse chromosome 16 (Mmu 16), that are homologous to genes that are harbored on Hsa21. A critical issue regarding the Ts65Dn model, however, is the fact that translocation of this minichromosome to the centromeric part of Mmu 17 is associated with triplication of 46 PCGs on Mmu17 that are not orthologs of Hsa21 (see [1,5,6]). It cannot be ruled out, therefore, that this may contribute to some of the phenotypes exhibited by the

<footnote><sup>a</sup> Department for Life Quality Studies (QUVI), University of Bologna, Corso D'Augusto 237 47921 Rimini, Italy</footnote>

<footnote><sup>b</sup> Department of Biomedical and Neuromotor Sciences (DIBINEM), University of Bologna, Piazza di Porta San Donato 2 40126 Bologna, Italy</footnote>

<footnote><sup>c</sup> Université de Strasbourg, CNRS, INSERM, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Department of Translational Medicine and Neurogenetics, 1 rue Laurent Fries 67404 Illkirch-Graffenstaden, France</footnote>

<footnote><sup>d</sup> Université de Strasbourg, CNRS, INSERM, CELPHEDIA, PHENOMIN-Institut Clinique de la Souris (ICS), 1 rue Laurent Fries 67404 Illkirch-Graffenstaden, France</footnote>

<footnote>* Corresponding authors: Renata Bartesaghi at Department of Biomedical and Neuromotor Sciences (DIBINEM), University of Bologna, Piazza di Porta San Donato 2, 40126 Bologna, Italy. Yann Herault at Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Department of Translational Medicine and Neurogenetics, 1 rue Laurent Fries, 67404 Illkirch-Graffenstaden, France.</footnote>

<footnote>E-mail addresses: herault@igbmc.fr (Y. Herault), renata.bartesaghi@unibo.it (R. Bartesaghi).</footnote>
Ts65Dn model. Due to this *caveat*, various other DS models have been created over the past 20 years (see [1,6]). These models replicate some DS phenotypes to various degrees, albeit, in most instances, to an extent that is less severe than in the Ts65Dn mouse. Considering that most of our knowledge regarding DS is based on evidence obtained in the Ts65Dn model, it is essential to establish the extent to which the triplication of extra genes contributes to its phenotypes. For this reason, the group led by Yann Herault has created a new model, the Ts66Yah mouse, derived from the Ts65Dn model. This new model is without the extra genes on Mmu 17 of the Ts65Dn mouse, which have been removed using CRISPR/Cas9 technology [7]. Phenotyping of this model in three independent laboratories revealed that it shows some behavioral defects that characterize the Ts65Dn mouse, even though variations may occur depending on the test used [7,8]. Similarly to the Ts65Dn model, this model exhibits age-related neuropathological changes in the hippocampus [9]. Gene expression analysis in hippocampal areas, however, shows that Ts66Yah mice exhibit alterations that only partially overlap with those exhibited by the Ts65Dn mouse [7].

Neurogenesis alterations and dendritic pathology appear very early in DS and are retained in adulthood; these neurodevelopmental defects are thought to critically underlie cognitive impairment (see [10]). As the Ts66Yah model has not been phenotypically characterized at early life stages, we deemed it important to establish whether it exhibits early impairment in neurogenesis and dendritogenesis similarly to individuals with DS. We focused on the hippocampus, a brain region whose development is severely impaired in people with DS, in the Ts65Dn mouse as well as in other DS models (see [11]). We examined these processes in Ts66Yah mice at two different moments during their life span, the neonatal period (15 days) and early adulthood (3 months). We chose these ages because our laboratory had previously demonstrated that at 15 days and at 3 months of age Ts65Dn mice exhibit impairment of hippocampal neurogenesis, dendritogenesis, and spinogenesis [12–16]. We found that at both ages Ts66Yah mice, unlike Ts65Dn mice, do not exhibit defects in dendritic maturation and neurogenesis. This suggests that mechanisms beyond neurogenesis and dendritogenesis may underpin the behavioral impairment seen in adulthood in the Ts66Yah model [7–9].

# 2. Materials and methods

## 2.1. Animals

Ts66Yah mice and wild-type (F1B6JC3B) mice were reared in the facility of the Institut de Génétique et de Biologie Moléculaire et Célulaire (IGBMC), Illkirch-Graffenstaden, France and were genotyped as previously described [7]. Male mice aged 15 days (P15 mice; Ts66Yah: n = 6; Wild type: n = 6) and three months (3 M mice; Ts66Yah: n = 6; Wild type: n = 6) were euthanized through cervical dislocation. Their brain was removed and cut across the midline. The right hemisphere was Golgi-stained, and the left hemisphere was fixed by immersion in 4 % paraformaldehyde in 100 mM phosphate buffer, stored in the fixative for 48 h, kept in 20 % sucrose in phosphate buffer for an additional 24–72 h and frozen at –80 °C. The left hemisphere was used for Ki-67 immunohistochemistry and DAPI-staining. All experiments were conducted following the ethical standards of French and European laws (European Communities Council Directive of 24 November 1986).

## 2.2. Histology

**Golgi staining.** The right hemisphere of P15 and 3 M mice was Golgi-stained using the FD Rapid Golgi Stain TM Kit (FD NeuroTechnologies, Inc.) as previously described [12]. The hemisphere was cut with a cryostat into 90-µm-thick coronal sections that were serially collected.

**Ki-67 immunohistochemistry.** The left hemisphere of P15 and 3 M mice was cut with a cryostat into 30-µm-thick coronal sections. One out of six sections from the hippocampal formation was mounted on slides and

immunostained for Ki-67 (diluted 1:100; ThermoFisher Scientific). Detection was performed using a Cy3-conjugated anti rabbit secondary antibody (diluted 1:200; Jackson Immunoresearch). Sections were counterstained with DAPI to label cell nuclei (Fig. 4A).

## 2.3. Measurements

### 2.3.1. Spine density

In Golgi-stained sections (Fig. 1A) from P15 and 3 M mice, spines of the granule cells of the dentate gyrus (DG) were counted live in 10 neurons per animal using a 100x oil immersion objective lens. Spines were sampled from dendritic segments in the inner and outer half (Fig. 1B) of the molecular layer (proximal dendrites and distal dendrites, respectively; Fig. 1C,D). For each neuron, we traced a line with a length of 20 µm over selected segments in the inner half (n = 3) and outer half (n = 3) of the molecular layer and counted all spines in each segment (Fig. 1E) using Image Pro Plus (Media Cybernetics, Silver Spring, MD 20910, USA) tools. The linear spine density was calculated by dividing the total number of spines counted by the length of the dendritic segment. Spine density was expressed as number of spines per 100 µm dendrite.

### 2.3.2. Measurement of the dendritic tree

The dendritic tree of the granule cells of P15 and 3 M mice was traced with dedicated custom-designed software (Immagini Computer di Maurizio Abbate & C. SNC, Bareggio, MI, Italy) interfaced with Image

<figure><img src="image_5.png" /></figure>

**Fig. 1.** Evaluation of spine density and dendritic arborization in Ts66Yah and euploid mice. (A) Low magnification image of a Golgi-stained coronal section across the hippocampal formation of a 3-month-old Ts66Yah mouse. Calibration bar = 1000 µm. (B) Higher magnification of the hippocampal dentate gyrus. Calibration bar = 100 µm. Examples of proximal (C) and distal (D) dendritic segments of the granule cells. Calibration bar = 10 µm. (E) High magnification image of distal dendrites of the granule cells. Spines (highlighted in red) were counted along a dendritic length of 20 µm (dotted line). (F) Example of the reconstructed dendritic tree of a granule neuron. Numbers indicate the different dendritic orders. Abbreviations: CA1, CA3, hippocampal fields; CX, cortex; dd, distal dendrites; DG, dentate gyrus; GR, granule cell layer; H, hilus; MOL, molecular layer; pd, proximal dendrites; TH, thalamus. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Pro Plus. The dendritic tree was traced live, at a final magnification of 500x, by focusing into the depth of the section. The operator starts with branches emerging from the cell soma and after having drawn the first parent branch goes on with all daughter branches of the next order in a centrifugal direction (Fig. 1F). At the end of tracing, the program reconstructs the total dendritic length, the number of branches of each order, and the mean length of branches of each order.

### 2.3.3. Count of Ki-67-positive cells

In P15 and 3 M mice, Ki-67-immunoreactive cells were quantified in every 6th coronal section of the DG spanning the entire anterior-posterior axis of the hippocampus (n = 17–22 sections). Quantification was carried out using a modified unbiased stereology protocol as previously described [15].

### 2.3.4. Number of granule cells in the DG

Unbiased stereology of the DG was performed in DAPI-stained sections of P15 and 3 M mice starting from a random position of the first (rostral most) section plane. The optical disector method was used to obtain cell density, and the Cavalieri principle was used to estimate volume, as previously described [17].

All measurements were carried out blind.

## 2.4. Statistics

Results are presented as mean ± standard error of the mean (SE). Data were analyzed using the IBM SPSS 22.0 software. Before running statistical analyses, we checked data distribution and homogeneity of variances for each variable using the Shapiro-Wilk test and Levene's test, respectively. If the data were normally distributed and variance was homogeneous, statistical analysis was carried out using the two-tailed Student's t-test. If the data were not normally distributed and variance was heterogeneous, transformations were made to achieve normality. If the transformed data did not achieve normality, statistical analysis was carried out using the Mann-Whitney U test. Based on the “Box plot” tool

available in SPSS Descriptive Statistics, in each analysis we excluded the extremes, i.e., values that were larger than 3 times the IQ range [$x \ge Q3 + 3 * (IQ)$; $x \le Q1-3 * (IQ)$]. A probability level of $p \le 0.05$ was considered to be statistically significant.

# 3. Results

## 3.1. Spine density in Ts66Yah and euploid mice

In Golgi-stained brains we evaluated spine density on both proximal and distal dendritic branches of the granule cells of the DG which are receptive fields of different inputs; the middle and outer thirds of the dendritic tree receive signals from the entorhinal cortex and the inner third is host to inputs from the mossy cells [18]. We found that P15 Ts66Yah mice had a similar spine density to that of euploid mice both on proximal and distal dendritic segments (Fig. 2A,B). Neither genotype showed any difference in spine density on proximal vs. distal dendritic branches. A comparison of spine density in 3 M mice showed that Ts66Yah mice had a similar spine density as their euploid counterparts on the distal dendrites (Fig. 2D) and a slightly (+9%) but significantly larger spine density on the proximal dendrites (Fig. 2C). In 3 M mice, spine density on the distal dendrites was significantly larger in comparison with spine density on the proximal dendrites both in euploid (about + 60 %) and Ts66Yah (about + 50 %) mice (Fig. 2C,D).

These results indicate that Ts66Yah mice, unlike Ts65Dn mice, exhibit no reduction in dendritic spine density either in the neonatal period or in early adulthood.

## 3.2. Dendritic arborization in Ts66Yah and euploid mice

We next evaluated the total dendritic length and number of branches (Fig. 1F) of the granule cells. In P15 Ts66Yah mice the dendritic arbor had a slightly reduced total length (−14%; Fig. 3A) in comparison with euploid mice but had a similar number of segments (Fig. 3B). To dissect possible effects on specific dendritic segments, we compared the length

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Dendritic spine density in the granule cells of Ts66Yah and euploid mice. Spine density on the proximal (A) and distal (B) dendrites of granule cells of euploid (n = 4) and Ts66Yah (n = 4) male mice aged 15 days (P15) and on the proximal (C) and distal (D) dendrites of granule cells of euploid (n = 6) and Ts66Yah (n = 6) male mice aged three months (3 M). Values in (A-D) represent mean ± SE. \* p < 0.05 (Ts66Yah vs. euploid mice); §§§ p < 0.001 (proximal vs. distal dendrites of the same genotype). Two-tailed *t*-test.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Dendritic arborization of the granule cells of Ts66Yah and euploid mice. Total dendritic length (A), total number of dendritic segments (B), mean length (C) and number (D) of branches of different orders of euploid (n = 4) and Ts66Yah (n = 5) male mice aged 15 days (P15). Total dendritic length (E), total number of dendritic segments (F), mean length (G), and number (H) of branches of different orders of euploid (n = 6) and Ts66Yah (n = 6) male mice aged three months (3 M). Values in (A-H) represent mean ± SE. (*) p = 0.054, * p < 0.05. Two-tailed t-test.</figcaption></figure>

and number of branches of each order in Ts66Yah vs. euploid mice. We found no difference between Ts66Yah and euploid mice in the length of branches of individual orders (Fig. 3C). Although branches of order 6 had a greater length in Ts66Yah vs. euploid mice this difference was only marginally significant (Fig. 3C). No difference was found in the mean number of branches of orders 1-6 (Fig. 3D). In 3 M Ts66Yah mice the dendritic arbor had a similar total length (Fig. 3E) and a similar number of segments (Fig. 3F) to that of euploid mice. We found no difference between Ts66Yah and euploid mice in the mean length (Fig. 3G) or number (Fig. 3H) of branches of each order. Similar to P15 Ts66Yah mice, in 3 M Ts66Yah mice the length of segments of the highest order (order 7) was larger in comparison with their euploid counterparts, but this difference was not statistically significant (Fig. 3G). Unlike branches of order 1-6, branches of order 7 were present in three mice per group only. Thus, the low number of cases may explain the lack of a statistically significant difference.

These results indicate that Ts66Yh mice, unlike Ts65Dn mice, exhibit almost no differences in the dendritic arborization of granule cells in comparison with their euploid counterparts. The only genotype effect involved the total dendritic length of P15 mice that was moderately shorter in Ts66Yah vs. euploid mice. This difference, however, was no

longer present in 3 M mice.

### 3.3. Proliferation potency of granule cell precursors of Ts66Yah and euploid mice

Ki-67 is an endogenous marker expressed by proliferating cells throughout almost the whole cell cycle. To establish possible genotype-related differences in the proliferation potency of granule cell precursors, we compared the total number of Ki-67-positive cells in the DG of Ts66Yah vs. euploid mice. An evaluation of the total number of Ki-67-positive cells showed no difference between Ts66Yah and euploid mice both at P15, a time at which hippocampal neurogenesis is very prominent [19], and 3 M (Fig. 4B,C), indicating no genotype effect on the proliferation potency of granule cell precursors.

### 3.4. Number of granule cells in the dentate gyrus of Ts66Yah and euploid mice

An evaluation of the stereology of the DG showed no differences between Ts66Yah and euploid mice in the volume of the granule cell layer, granule cell density and total number of granule cells both in P15
<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Stereology of the dentate gyrus of Ts66Yah and euploid mice. (A) Sections across the dentate gyrus of a euploid and a Ts66Yah mouse aged 15 days. Sections were immunostained for Ki-67 and counterstained with DAPI. The insets represent examples of Ki-67-positive cells (red cells). (B,C) Total number of Ki-67-positive cells in the granule cell layer of the dentate gyrus of euploid (n = 4) and Ts66Yah (n = 5) male mice aged 15 days (B) and euploid (n = 4) and Ts66Yah (n = 4) mice aged 3 months (C). (D-F) Volume of the granule cell layer (D), granule cell density (E), and total number of granule cells (F) in the dentate gyrus of euploid (n = 4) and Ts66Yah (n = 5) male mice aged 15 days; same mice as in (B). (G-I) Volume of the granule cell layer (G), granule cell density (H), and total number of granule cells (I) in the dentate gyrus of euploid (n = 4) and Ts66Yah (n = 4) male mice aged 3 months; same mice as in (C). Values in (B-I) represent mean ± SE. Calibration bars: low magnification = 100 µm; high magnification = 10 µm. Abbreviations: GR, granule cell layer; H, hilus; MOL, molecular layer. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

(Fig. 4D-F) and 3 M (Fig. 4G-I) mice. These results are consistent with the absence of differences in the proliferation potency exhibited by the precursors of the granule cells of Ts66Yah vs. euploid mice.

# 4. Discussion

## 4.1. Lack of dendritic pathology and neurogenesis alterations in the Ts66Yah model

We found no reduction in spine density on the granule cell dendrites of Ts66Yah mice compared to their euploid counterparts. This contrasts with the spine density reduction that characterizes hippocampal and cortical neurons of Ts65Dn mice [12–15,20–23] as well as Ts1Cje, Ts1Rhr, and Dp16 mice [24–26]. A difference in spine density in Ts66Yah vs. euploid mice was only present on the proximal dendrites of 3 M mice (Fig. 2C). Still, it went in a direction opposite (i.e. more spines in Ts66Yah vs. euploid mice) to that seen in Ts65Dn and other DS models. Regarding dendritic arborization, we found that the granule cells of Ts66Yah mice had a dendritic arbor that was substantially similar to that of their euploid counterparts. The only difference was a moderate reduction in total dendritic length in P15 Ts66Yah, which disappeared at three months. These results are at variance with the dendritic hypotrophy that characterizes hippocampal and cortical neurons of Ts65Dn mice at juvenile and adult ages [12–15,23,27–29]. Regarding hippocampal neurogenesis, Ts66Yah mice had a similar number of granule cell precursors and total number of granule neurons to those of their euploid counterparts, indicating no impairment of

hippocampal neurogenesis. These results are at variance with ample evidence showing reduced neurogenesis, cellularity, and volume in the dentate gyrus of Ts65Dn mice (reviewed by [30]), reduced cellularity in the dentate gyrus of Dp1Tyb mice [31], and impaired neurogenesis in Ts1Cje and Ts2Cje mice [32–34]. Altogether the lack of early neurogenesis changes in the Ts66Yah model may reflect the lack of neurogenesis changes observed in the fetal Dp(16Lipi-Zbtb21)1Yey model [35].

The Ts66Yah model contains the same genetic composition as Ts65Dn mice except for the absence of 46 PCGs that are non-DS-related. The lack of dendritic pathology and neurogenesis impairment in Ts66Yah mice might suggest that in Ts65Dn mice all these defects are due to the extra genes on Mmu17. The Ts1Cje and Ts1Rhr models of DS are trisomic for genes (~70 and ~30 genes, respectively) that cover part of the trisomic region of Ts65Dn mice in different genetic backgrounds but bear no triplication of extra genes (see [6]). Ts1Cje and Ts1Rhr mice exhibit a reduced spine density [24,25] and Ts1Cje mice also exhibit proliferation impairment, though to a milder degree in comparison with Ts65Dn mice [32–34], suggesting that the triplicated genes on Mmu16 shared by Ts65Dn, Ts1Cje, and Ts1Rhr mice are sufficient to impair dendritic development and neurogenesis. Another hypothesis is that a multigenic contribution to the phenotypes, with positive and negative gene dosage contribution, may underlie the differences between Ts66Yah mice and Ts65Dn mice, due to trisomy of the Mmu17 centromeric region, as well as Ts1Cje and Ts1Rhr mice. Further investigations with other partial duplication models are required to shed light on these differences and their root cause [36].
The Ts65Dn mouse was generated by irradiation of DBA/2J mice, which were crossed with C57BL/6J to recover the radiation-induced minichromosome, and the stocks carrying translocations of interest were maintained on a F1B6C3H genetic background [37]. It has been shown that the alleles of *Ezrin*, which is one of the extra genes on Mmu17, and which may be involved in spinogenesis [38,39], are similar in the DBA/2J and C3He/HeJ genetic backgrounds but are different in the C57BL/6J background (the background of Ts1Cje and Ts1Rhr mice) [40]. While differences in the genetic background may contribute to differences in the severity of phenotypes across models, the lack of spinogenesis, dendritogenesis, and neurogenesis defects in Ts66Yah mice remains puzzling. A possible explanation is that deletion of the extra genes on Mmu17 causes complex modifications in gene expression that affect the increased ambulatory activity found in Ts65Dn and not in Ts66Yah mice, that hamper the emergence of the neurodevelopmental phenotypes exhibited by Ts65Dn mice, or reduce their penetrance and expressivity at early life stages. Indeed, gene expression profiling in the adult hippocampal region showed differences between Ts66Yah and Ts65Dn mice in the expression of genes harbored not only on Mmu16 but also on various other chromosomes [7]. The Ts66Yah mouse line was generated using CRISPR/Cas9 technology based on the Ts65Dn 1924 line obtained from the Jackson laboratory [7]. A thorough phenotypic analysis of the 1924 line (as well as of the 5252 line) starting from the year 2010 has revealed the disappearance or attenuation of numerous phenotypes over time [41]. The founder that was used to create the Ts66Yah line was derived from a Ts65Dn stock imported before 2010 in the laboratory of Yann Hérault [7]. The stock was used in several studies and showed a large spectrum of behavioral phenotypes [7,42,43]. Although it seems improbable that this colony had an attenuated phenotype, this possibility cannot be ruled out.

Creating DS models that mimic the whole gamut of DS phenotypes still represents a challenge for several reasons. DS is a contiguous gene syndrome, spanning the entire Hsa21, while the orthologs of Hsa21 genes are present on segments of three mouse chromosomes (Mmu16, Mmu17, and Mmu10). Triplicated genes in DS are harbored in a freely segregating chromosome (aneuploidy), which is the case for Ts65Dn mice but not most DS models. These problems may explain the differences in different DS models and lack of DS-related phenotypes exhibited by some of them. The creation of the Ts66Yah has been important because these mice do not bear triplication of DS-unrelated genes. Yet, although Ts66Yah mice display learning deficits, they do not exhibit the defects in hippocampal development (current study) or other early neurodevelopmental defects [44] that are typical of DS. While additional studies are needed to characterize the phenotype of Ts66Yah mice further, the current results suggest that this model is unsuitable for studying the long-term neurodevelopmental phenotypes that characterize DS. In order to examine neurodevelopmental changes that are more closely related to DS, additional models, with all the regions homologous to human chromosome 21 triplicated, for example, should be considered.

### CRediT authorship contribution statement

**Marco Emili:** Methodology, Investigation, Formal analysis. **Fiorenza Stagni:** Investigation, Formal analysis, Conceptualization. **Sandra Guidi:** Investigation, Formal analysis, Conceptualization. **Carla Russo:** Investigation. **Claire Chevalier:** Investigation. **Arnaud Duchon:** Investigation. **Yann Herault:** Writing – review & editing, Writing – original draft, Conceptualization. **Renata Bartesaghi:** Writing – review & editing, Writing – original draft, Conceptualization.

### Author contribution

Conceptualization: FS, RB, SG and YH; Rearing of mice, preparation of samples: AD and CC; Evaluation of dendritic arborization: ME; Evaluation of dendritic spines: FS and SG; Histology: CR and ME; Stereology:

CR and RB; Statistical analysis: FS, ME and SG; Writing and original draft preparation: RB and YH. All authors have read and agreed to the published version of the manuscript.

### Funding

This work was supported by grants to Y.H. [the French state funds through the Agence Nationale de la Recherche under the project PRC DYRK-DOWN ANR-18-CE16-0020, and support from program Investissements d'Avenir labeled IdEx Unistra (ANR-10-IDEX-0002), a SFRI-STRAT'US project (ANR 20-SFRI-0012), EUR IMCBio (ANR-17-EURE-0023), INBS PHENOMIN (ANR-10-INBS-07 PHENOMIN), and the Foundation Jerome Lejeune, and to R. B. ("Fondazione Generali e Assicurazione Generali", Italy).

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Data availability

Data will be made available on request.

### References

[1] M.D.M. Muñiz Moreno, V. Brault, M.C. Birling, G. Pavlovic, Y. Herault, Modeling Down syndrome in animals from the early stage to the 4.0 models and next, Prog. Brain Res. 251 (2020) 91–143, https://doi.org/10.1016/bs.pbr.2019.08.001.

[2] M.J. Bull, Down syndrome, N. Engl. J. Med. 382 (2020) 2344–2352, https://doi.org/10.1056/NEJMra1706537.

[3] J. Grieco, M. Pulsifer, K. Seligsohn, B. Skotko, A. Schwartz, Down syndrome: Cognitive and behavioral functioning across the lifespan, Am. J. Med. Genet. C Semin. Med. Genet. 169 (2015) 135–149, https://doi.org/10.1002/ajmgc.31439.

[4] M.T. Davisson, C. Schmidt, R.H. Reeves, N.G. Irving, E.C. Akeson, B.S. Harris, R. T. Bronson, Segmental trisomy as a mouse model for Down syndrome, Prog. Clin. Biol. Res. 384 (1993) 117–133.

[5] A. Duchon, M. Raveau, C. Chevalier, V. Nalesso, A.J. Sharp, Y. Herault, Identification of the translocation breakpoints in the Ts65Dn and Ts1Cje mouse lines: relevance for modeling Down syndrome, Mamm. Genome 22 (2011) 674–684, https://doi.org/10.1007/s00335-011-9356-0.

[6] Y. Herault, J.M. Delabar, E.M.C. Fisher, V.L.J. Tybulewicz, E. Yu, V. Brault, Rodent models in Down syndrome research: impact and future opportunities, Dis. Model. Mech. 10 (2017) 1165–1186, https://doi.org/10.1242/dmm.029728.

[7] A. Duchon, M. Del Mar Muñiz, C. Moreno, V. Chevalier, P. Nalesso, M. Andre, M. Fructuoso-Castellar, C. Mondino, V. Po, M.C. Noblet, M.C. Birling, Y.H. Potier, Ts66Yah, a mouse model of Down syndrome with improved construct and face validity, Dis. Model. Mech. 15 (2022), https://doi.org/10.1242/dmm.049721.

[8] F. Guedj, E. Kane, L.A. Bishop, J.L.A. Pennings, Y. Herault, D.W. Bianchi, The impact of Mmu17 Non-Hsa21 orthologous genes in the Ts65Dn mouse model of down syndrome: The gold standard refuted, Biol. Psychiatry 94 (2023) 84–97, https://doi.org/10.1016/j.biopsych.2023.02.012.

[9] C. Lanzillotta, M.R. Baniowska, F. Prestia, C. Sette, V. Nalesso, M. Perluigi, E. Barone, A. Duchon, A. Tramutola, Y. Herault, F. Di Domenico, Shaping down syndrome brain cognitive and molecular changes due to aging using adult animals from the Ts66Yah murine model, Neurobiol. Dis. 196 (2024) 106523, https://doi.org/10.1016/j.nbd.2024.106523.

[10] R. Bartesaghi, S. Vicari, W.C. Mobley, Prenatal and postnatal pharmacotherapy in down syndrome: the search to prevent or ameliorate neurodevelopmental and neurodegenerative disorders, Annu. Rev. Pharmacol. Toxicol. 62 (2022) 211–233, https://doi.org/10.1146/annurev-pharmtox-041521-103641.

[11] F. Stagni, R. Bartesaghi, The challenging pathway of treatment for neurogenesis impairment in down syndrome: achievements and perspectives, Front. Cell. Neurosci. 16 (2022) 903729, https://doi.org/10.3389/fncel.2022.903729.

[12] S. Guidi, F. Stagni, P. Bianchi, E. Ciani, E. Ragazzi, S. Trazzi, G. Grossi, C. Mangano, L. Calza, R. Bartesaghi, Early pharmacotherapy with fluoxetine rescues dendritic pathology in the Ts65Dn mouse model of Down syndrome, Brain Pathol. 23 (2013) 129–143, https://doi.org/10.1111/j.1750-3639.2012.00624.x.

[13] F. Stagni, A. Giacomini, S. Guidi, E. Ciani, E. Ragazzi, M. Filonzi, R. De Iasio, R. Rimondini, R. Bartesaghi, Long-term effects of neonatal treatment with fluoxetine on cognitive performance in Ts65Dn mice, Neurobiol. Dis. 74C (2015) 204–218, https://doi.org/10.1016/j.nbd.2014.12.005.

[14] F. Stagni, A. Giacomini, M. Emili, S. Trazzi, S. Guidi, M. Sassi, E. Ciani, R. Rimondini, R. Bartesaghi, Short- and long-term effects of neonatal pharmacotherapy with epigallocatechin-3-gallate on hippocampal development in the Ts65Dn mouse model of Down syndrome, Neuroscience 333 (2016) 277–301, https://doi.org/10.1016/j.neuroscience.2016.07.031.
[15] F. Stagni, A. Giacomini, S. Guidi, M. Emili, B. Uguagliati, M.E. Salvalai, V. Bortolotto, M. Grilli, R. Rimondini, R. Bartesaghi, A flavonoid agonist of the TrkB receptor for BDNF improves hippocampal neurogenesis and hippocampus-dependent memory in the Ts65Dn mouse model of DS, Exp. Neurol. 298 (2017) 79–96, https://doi.org/10.1016/j.expneurol.2017.08.018.

[16] P. Bianchi, E. Ciani, S. Guidi, S. Trazzi, D. Felice, G. Grossi, M. Fernandez, A. Giuliani, L. Calza, R. Bartesaghi, Early pharmacotherapy restores neurogenesis and cognitive performance in the Ts65Dn mouse model for Down syndrome, *Neurosci. 30* (2010) 8769–8779.

[17] F. Stagni, M.E. Salvalai, A. Giacomini, M. Emili, B. Uguagliati, E. Xia, M. Grilli, R. Bartesaghi, S. Guidi, Neonatal treatment with cyclosporine A restores neurogenesis and spinogenesis in the Ts65Dn model of Down syndrome, Neurobiol. Dis. 129 (2019) 44–55, https://doi.org/10.1016/j.nbd.2019.05.005.

[18] D.G. Amaral, M.P. Witter, Hippocampal Formation: The Rat Nervous System, Academic Press, 1995, pp. 443–493.

[19] J. Altman, S. Bayer, Postnatal development of the hippocampal dentate gyrus under normal and experimental conditions, in: R.L. Isaacson, K.H. Pribram (Eds.), *The Hippocampus*, 95–122, Plenum Press, New York and London, p 1975, pp. 95–122.

[20] P.V. Belichenko, E. Masliah, A.M. Kleschevnikov, A.J. Villar, C.J. Epstein, A. Salehi, W.C. Mobley, Synaptic structural abnormalities in the Ts65Dn mouse model of Down Syndrome, J Comp Neurol 480 (2004) 281–298, https://doi.org/10.1002/cne.20337.

[21] S. Catuara-Solarz, J. Espinosa-Carrasco, I. Erb, K. Langohr, J.R. Gonzalez, C. Notredame, M. Dierssen, Combined treatment with environmental enrichment and (-)-epigallocatechin-3-gallate ameliorates learning deficits and hippocampal alterations in a mouse model of down syndrome, eNeuro 3 (2016), https://doi.org/10.1523/ENEURO.0103-16.2016.

[22] R. Benavides-Piccione, I. Ballesteros-Yanez, M.M. de Lagran, G. Elston, X. Estivill, C. Fillat, J. Defelipe, M. Dierssen, On dendrites in Down syndrome and DS murine models: a spiny way to learn, Prog. Neurobiol. 74 (2004) 111–126, https://doi.org/10.1016/j.pneurobio.2004.08.001.

[23] M. Dierssen, R. Benavides-Piccione, C. Martinez-Cue, X. Estivill, J. Florez, G. N. Elston, J. DeFelipe, Alterations of neocortical pyramidal cell phenotype in the Ts65Dn mouse model of Down syndrome: effects of environmental enrichment, Cereb. Cortex 13 (2003) 758–764, https://doi.org/10.1093/cercor/13.7.758.

[24] P.V. Belichenko, A.M. Kleschevnikov, A. Salehi, C.J. Epstein, W.C. Mobley, Synaptic and cognitive abnormalities in mouse models of Down syndrome: exploring genotype-phenotype relationships, J Comp Neurol 504 (2007) 329–345, https://doi.org/10.1002/cne.21433.

[25] N.P. Belichenko, P.V. Belichenko, A.M. Kleschevnikov, A. Salehi, R.H. Reeves, W. C. Mobley, The "Down syndrome critical region" is sufficient in the mouse model to confer behavioral, neurophysiological, and synaptic phenotypes characteristic of Down syndrome, J Neurosci. 29 (2009) 5938–5948, https://doi.org/10.1523/JNEUROSCI.1547-09.2009.

[26] B. Pinto, G. Morelli, M. Rastogi, A. Savardi, A. Fumagalli, A. Petretto, M. Bartolucci, E. Varea, T. Catelani, A. Contestabile, L.E. Perlini, L. Cancedda, Rescuing over-activated microglia restores cognitive performance in juvenile animals of the Dp(16) mouse model of down syndrome, Neuron 108 (2020) 887–904.e12, https://doi.org/10.1016/j.neuron.2020.09.010.

[27] B. Uguagliati, F. Stagni, M. Emili, A. Giacomini, C. Russo, S. Guidi, R. Bartesaghi, Early appearance of dendritic alterations in neocortical pyramidal neurons of the Ts65Dn model of down syndrome, Dev. Neurosci. 44 (2022) 23–38, https://doi.org/10.1159/000520925.

[28] V. Dang, B. Medina, D. Das, S. Moghadam, K.J. Martin, B. Lin, P. Naik, D. Patel, R. Nosheny, J. Wesson Ashford, A. Salehi, Formoterol, a long-acting beta2 adrenergic agonist, improves cognitive function and promotes dendritic complexity in a mouse model of Down syndrome, Biol. Psych. 75 (2014) 179–188, https://doi.org/10.1016/j.biopsych.2013.05.024.

[29] J. Carbonell, J.M. Blasco-Íbáñez, C. Crespo, J. Nácher, E. Varea, Piriform cortex alterations in the Ts65Dn model for down syndrome, Brain Res. 1747 (2020) 147031, https://doi.org/10.1016/j.brainres.2020.147031.

[30] F. Stagni, A. Giacomini, M. Emili, S. Guidi, R. Bartesaghi, Neurogenesis impairment: An early developmental defect in Down syndrome, Free Radic. Biol. Med. 114 (2018) 15–32, https://doi.org/10.1016/j.freeradbiomed.2017.07.026.

[31] M.E. Serrano, E. Kim, B. Siow, D. Ma, L. Rojo, C. Simmons, D. Hayward, D. Gibbs, N. Singh, A. Strydom, E.M.C. Fisher, V.L.J. Tybulewicz, D. Cash, Investigating brain alterations in the Dp1Tyb mouse model of Down syndrome, Neurobiol. Dis. 188 (2023) 106336, https://doi.org/10.1016/j.nbd.2023.106336.

[32] R.X. Moldrich, L. Dauphinot, L. Laffaire, T. Vitalis, Y. Herault, P.M. Beart, J. Rossier, D. Vivien, C. Gehrig, S.E. Antonarakis, R. Lyle, M.C. Potier, Proliferation deficits and gene expression dysregulation in Down's syndrome (Ts1Cje) neural progenitor cells cultured from neurospheres, J. Neurosci. Res. 87 (2009) 3143–3152, https://doi.org/10.1002/jnr.22131.

[33] C.A. Hewitt, K.H. Ling, T.D. Merson, K.M. Simpson, M.E. Ritchie, S.L. King, M. A. Pritchard, G.K. Smyth, T. Thomas, H.S. Scott, A.K. Voss, Gene network disruptions and neurogenesis defects in the adult Ts1Cje mouse model of Down syndrome, PLoS One 5 (2010) e11561, https://doi.org/10.1371/journal.pone.0011561.

[34] K. Ishihara, K. Amano, E. Takaki, A. Shimohata, H. Sago, C.J. Epstein, K. Yamakawa, Enlarged brain ventricles and impaired neurogenesis in the Ts1Cje and Ts2Cje mouse models of Down syndrome, Cereb. Cortex 20 (2010) 1131–1143, https://doi.org/10.1093/cercor/bhp176.

[35] J.W. Goodliffe, J.L. Olmos-Serrano, N.M. Aziz, J.L. Pennings, F. Guedj, D. W. Bianchi, T.F. Haydar, Absence of prenatal forebrain defects in the Dp(16)1Yey/+ mouse model of down syndrome, J. Neurosci. 36 (2016) 2926–2944, https://doi.org/10.1523/JNEUROSCI.2513-15.2016.

[36] A. Duchon, M. Del Mar Muniz, S.M. Moreno, M.P.S. Lorenzo, C. de Souza, V. Chevalier, H. Nalesso, P.L. Meziane, V. de Sousa, J.P. Noblet, V. Armaspach, Y. H. Brault, Multi-influential genetic interactions alter behaviour and cognition through six main biological cascades in Down syndrome mouse models, Hum. Mol. Genet. 30 (2021) 771–788, https://doi.org/10.1093/hmg/ddab012.

[37] M.T. Davisson, C. Schmidt, E.C. Akeson, Segmental trisomy of murine chromosome 16: a new model system for studying Down syndrome, Prog. Clin. Biol. Res. 360 (1990) 263–280.

[38] J.J. Jayasundar, J.H. Ju, L. He, D. Liu, F. Meilleur, J. Zhao, D.J. Callaway, Z. Bu, Open conformation of ezrin bound to phosphatidylinositol 4,5-bisphosphate and to F-actin revealed by neutron scattering, J. Biol. Chem. 287 (2012) 37119–37133, https://doi.org/10.1074/jbc.JBC.M112.380972.

[39] Y. Furutani, M. Kawasaki, H. Matsuno, S. Mitsui, K. Mori, Y. Yoshihara, Vitronectin induces phosphorylation of ezrin/radixin/moesin actin-binding proteins through binding to its novel neuronal receptor telencephalin, J. Biol. Chem. 287 (2012) 39041–39049, https://doi.org/10.1074/jbc.M112.383851.

[40] J.A. Blake, R. Baldarelli, J.A. Kadin, J.E. Richardson, C.L. Smith, C.J. Bult, Mouse genome database (MGD): Knowledgebase for mouse-human comparative biology, Nucleic Acids Res. 49 (2021) D981–D987, https://doi.org/10.1093/nar/gkaa1083.

[41] P.R. Shaw, J.A. Klein, N.M. Aziz, T.F. Haydar, Longitudinal neuroanatomical and behavioral analyses show phenotypic drift and variability in the Ts65Dn mouse model of Down syndrome, Dis. Model. Mech. 13 (2020), https://doi.org/10.1242/dmm.046243.

[42] R. De la Torre, S. De Sola, M. Pons, A. Duchon, M.M. de Lagran, M. Farre, M. Fito, B. Benejam, K. Langohr, J. Rodriguez, M. Pujadas, J.C. Bizot, A. Cuenca, N. Janel, S. Catuara, M.I. Covas, H. Blehaut, Y. Herault, J.M. Delabar, M. Dierssen, Epigallocatechin-3-gallate, a DYRK1A inhibitor, rescues cognitive deficits in Down syndrome mouse models and in humans, Mol. Nutr. Food Res. 58 (2014) 278–288, https://doi.org/10.1002/mnfr.201300325.

[43] T.L. Nguyen, A. Duchon, A. Manousopoulou, N. Loaec, B. Villiers, G. Pani, M. Karatas, A.E. Mechling, L.A. Harsan, E. Limanton, J.P. Bazineau, F. Carreaux, S. D. Garbis, L. Meijer, Y. Herault, Correction of cognitive deficits in mouse models of Down syndrome by a pharmacological inhibitor of DYRK1A, Dis. Model. Mech. 11 (2018), https://doi.org/10.1242/dmm.035634.

[44] P.R. Shaw, Identifying Phenotypic Change Across Time In Mouse Models of Down Syndrome, Boston University Theses & Dissertations [9340], 2021. https://hdl.handle.net/2144/42320,.