Neurobiology of Disease 74 (2015) 204–218

Contents lists available at ScienceDirect

Neurobiology of Disease

j ourna l homepage: www.e lsev ie r .com/ locate /ynbd i

Long-term effects of neonatal treatment with fluoxetine on cognitiveperformance in Ts65Dn mice☆

Fiorenza Stagni a, Andrea Giacomini a, Sandra Guidi a, Elisabetta Ciani a, Elena Ragazzi a, Mirco Filonzi b,Rosaria De Iasio b, Roberto Rimondini c, Renata Bartesaghi a,⁎a Department of Biomedical and Neuromotor Sciences, University of Bologna, Italyb Centralized Laboratory, S. Orsola-Malpighi University Hospital, Bologna, Italyc Department of Medical and Surgical Sciences, University of Bologna, Italy

☆ Authorship note: Fiorenza Stagni, Andrea Giacominequally to this work.⁎ Corresponding author at: Department of Biomedi

Physiology Building, Piazza di Porta San Donato 2, I-401051 2091737.

E-mail address: renata.bartesaghi@unibo.it (R. BartesaAvailable online on ScienceDirect (www.sciencedir

http://dx.doi.org/10.1016/j.nbd.2014.12.0050969-9961/© 2014 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 22 June 2014Revised 18 November 2014Accepted 1 December 2014Available online 10 December 2014

Keywords:Down syndromeCognitive impairmentPharmacotherapy5-HT1A receptorAlzheimer's disease

Individuals with Down syndrome (DS), a genetic condition caused by triplication of chromosome 21, are charac-terized by intellectual disability and are prone to develop Alzheimer's disease (AD), due to triplication of theamyloid precursor protein (APP) gene. Recent evidence in the Ts65Dn mouse model of DS shows that enhance-ment of serotonergic transmission with fluoxetine during the perinatal period rescues neurogenesis, dendriticpathology and behavior, indicating that cognitive impairment can be pharmacologically restored. A crucialquestion is whether the short-term effects of early treatments with fluoxetine disappear at adult life stages. Inthe current study we found that hippocampal neurogenesis, dendritic pathology and hippocampus/amygdala-dependent memory remained in their restored state when Ts65Dn mice, which had been neonatally treatedwith fluoxetine, reached adulthood. Additionally, we found that the increased levels of the APP-derived βCTFpeptide in adult Ts65Dn mice were normalized following neonatal treatment with fluoxetine. This effect wasaccompanied by restoration of endosomal abnormalities, aβCTF-dependent feature of DS and AD.While untreatedadult Ts65Dnmice had reduced hippocampal levels of the 5-HT1A receptor (5-HT1A-R) and methyl-CpG-bindingprotein (MeCP2), a protein that promotes 5-HT1A-R transcription, in neonatally-treated mice both 5-HT1A-R andMeCP2 were normalized. In view of the crucial role of serotonin in brain development, these findings suggest thatthe enduring outcome of neonatal treatment with fluoxetine may be due to MeCP2-dependent restoration of the5-HT1A-R. Taken together, results provide new hope for the therapy of DS, showing that early treatment withfluoxetine enduringly restores cognitive impairment and prevents early signs of AD-like pathology.

© 2014 Elsevier Inc. All rights reserved.

Introduction

Intellectual disability is the invariable hallmark and the mostinvalidating feature of Down syndrome (DS), a genetic disorder causedby triplication of chromosome 21. Moreover, individuals with DS areprone to develop Alzheimer's disease (AD) in adulthood, with conse-quent worsening of cognitive functions (Dierssen, 2012). Due to animprovement in medical care, life expectancy for individuals with DShas increased during the last decade and, consequently, individualswith DS may outlive their parents.

Intellectual impairment in DS is due to alterations of brain develop-ment that can be traced back to fetal life stages. These alterations

i and Sandra Guidi contributed

cal and Neuromotor Sciences,26 Bologna BO, Italy. Fax: +39

ghi).ect.com).

include widespread neurogenesis impairment, excessive number ofastrocytes, dendritic atrophy, and connectivity impairment (Bartesaghiet al., 2011). These defects are distributed throughout the brain and,therefore, numerous brain functions are altered. Much effort has beentaken during the past decade to identify the gene/s responsible for theaberrant brain development and cognitive profile of DS. The geneticculprits have not been unequivocally identified, most likely becausethese alterations do not depend on a single gene but rather, derivefrom a mixture of actions, to which various genes may contribute.Nevertheless, candidate genes that play an important role in bothbrain development and function can be used as targets for therapeuticinterventions. In addition, therapies can be attempted by acting down-stream, seeking to improve structural and neurochemical defectsengendered by gene triplication.

Various pharmacological approaches have been attempted inmousemodels of DS, showing that it is possible to improve some of themorpho-functional defects of the trisomic brain and related behavior(Bartesaghi et al., 2011; Costa and Scott-McKean, 2013). Most of thesestudies have been carried out in adult animals. It must be noted,

205F. Stagni et al. / Neurobiology of Disease 74 (2015) 204–218

however, that in spite of a certain degree of plasticity retained in adult-hood, the overall organization of the brain mainly depends on eventstaking place during embryonic and perinatal life stages. Consideringthat one of the most severe defects of trisomy is early neurogenesisimpairment (Guidi et al., 2008; Guidi et al., 2011), therapies to improvethis defect should be started as soon as possible, otherwise the brainwillhave a permanently reduced asset of neurons. Likewise, the severedendritic pathology due to trisomy should be restored as early as possi-ble, in order to allow the establishment of appropriate neuronal connec-tions. With this idea in mind, we attempted to rescue the morpho-functional defects of the trisomic brain with early pharmacotherapies.We decided to use fluoxetine, a selective serotonin reuptake inhibitorbecause: 1) the serotonergic system is altered in DS (Bar-Peled et al.,1991; Boullin and O'Brien, 1971; Lott et al., 1972a, 1972b; Whittleet al., 2007); 2) serotonin is fundamental for neurogenesis, dendriticdevelopment and synaptogenesis (Banasr et al., 2004; Encinas et al.,2006; Mogha et al., 2012; Santarelli et al., 2003; Whitaker-Azmitia,2001). We found that treatment with fluoxetine during the embryonicperiod rescued overall brain development and that neonatal treatmentinduced full recovery of hippocampal neurogenesis, dendritic develop-ment, connectivity and hippocampus-dependent memory. The effectof either the pre- or early postnatal treatments were retained up toadolescence (Bianchi et al., 2010; Guidi et al., 2014; Guidi et al., 2013).

While these results clearly show that it is possible to rescue themajor brain defects caused by gene triplication with early interventionand that this effect can outlast treatment cessation, there is a crucialquestion that still needs an answer. Do the restoring effects of an earlytherapy vanish in the long run or is the rescue of development a long-term outcome that extends to adult life stages? The question is not triv-ial because fluoxetine (or other drugs) do not reduce the gene burdenbut simply bypass it, by interfering with its effects. Consequently, thepossibility exists that an initial restoration of brain development maygo awry with time, once the therapy is interrupted. Therefore, the goalof the current studywas to establishwhether an early pharmacotherapywith fluoxetine has long-term effects on the brain that are retained atadult life stages. To this purpose, we treated neonate Ts65Dn micewith fluoxetine during the first two postnatal weeks and examinedthe outcome of treatment when the same mice reached adulthood.

Materials and methods

Colony

Female Ts65Dn mice carrying a partial trisomy of chromosome16 (Reeves et al., 1995) were obtained from Jackson Laboratories(Bar Harbour, ME, USA) and the original genetic backgroundwas main-tained by mating them with C57BL/6JEi x C3H/HeSnJ (B6EiC3Sn) F1males. Animals were karyotyped as previously described (Reinholdtet al., 2011). The day of birth was designated postnatal day zero. Atotal of 77 mice were used. The animals' health and comfort werecontrolled by the veterinary service. The animals had access to waterand food ad libitum and lived in a room with a 12:12 h dark/lightcycle. Experiments were performed in accordance with the Italian andEuropean Community law for the use of experimental animals andwere approved by Bologna University Bioethical Committee. In thisstudy all efforts were made to minimize animal suffering and to keepthe number of animals used to a minimum.

Experimental protocol

Euploid (n= 23) and Ts65Dn (n= 12)mice received a daily subcu-taneous injection (at 9–10 a.m.) of fluoxetine (Sigma-Aldrich) in0.9% NaCl solution from postnatal day 3 (D3) to D15 (dose: 5 mg/kgfrom D3 to D7; 10 mg/kg from D8 to D15). Age-matched euploid(n = 22) and Ts65Dn (n = 20) mice were injected with the vehicle(Fig. 1). The total number of Ts65Dn mice used was smaller in

comparison with that of euploid mice because the number of trisomicpups in a litter is only approximately 20–40% (Roper et al., 2006). Thesmaller number of treated Ts65Dn mice in comparison with theiruntreated counterparts (see above)was not due to a treatment-inducedhigh mortality rate but to the fact that, for unpredictable reasons, in thelitters treated with fluoxetine the number of trisomic pups was smallerin comparison with that in untreated litters. Each treatment group hadapproximately the same composition of males and females. Animalswere killed at the age of 3.0–3.5months and bodyweight was recordedprior to sacrifice. After sacrifice, the brain was excised and weighed.Data on body and brain weight are reported in Supplementary results.Starting from eighteen days before reaching 3.0–3.5 months of agemice were behaviorally tested (Fig. 1). Some of these animals (5–6animals for each condition) received an intraperitoneal injection(150 μg/g body weight) of BrdU (5-bromo-2-deoxyuridine; Sigma),a marker of proliferating cells (Nowakowski et al., 1989) in 0.9% NaClsolution (at 11–12 a.m.) one month before being killed (Fig. 1). Thebrain of another group of animals (5–8 animals for each condition)was quickly removed, the hippocampal formation and a 2–3 mm-thick brain slice immediately rostral to the hippocampal formationthat included the septal region (called here septal forebrain) weredissected, kept at −80 °C and used for Western blotting. A summaryof the overall study design is reported in Supplementary Table 1.

Histological procedures

Mice that had received BrdU were deeply anesthetized, the brainwas removed and the left hemisphere was fixed by immersion in 4%paraformaldehyde in 100 mM phosphate buffer, pH 7.4. These hemi-sphereswere stored in the fixative for 72 h, kept in 20% sucrose in phos-phate buffer for an additional 24 h, frozen and stored at -80 °C. The righthemisphere was rinsed in PBS and Golgi-stained. The left hemispherewas cut with a freezing microtome into 30-μm-thick coronal sectionsthat were serially collected in antifreezing solution and used for thefollowing procedures (see also Table 1): immunohistochemistry fori) Ki-67, ii) cleaved caspase-3, iii) DCX, iv) SYN, and v) Rab5 anddouble-fluorescence immunohistochemistry for BrdU and either aneuronal (NeuN) or an astrocytic (GFAP) marker. Details of histologicalprocedures and related measurements are reported in Supplementarymethods.

Western blotting

Total proteins were obtained as previously described (Trazzi et al.,2011). We analyzed the levels of p21, 5-HT1A receptor (5-HT1A-R),Erk1/2 and phospho-Erk1/2, methyl-CpG-binding protein (MeCP2),brain-derived neurotrophic factor (BDNF), amyloid precursor protein(APP), APP carbossiterminal fragment beta (βCTF) and beta site cleav-ing enzyme 1 (BACE1) in homogenates of the hippocampal formationand the levels of 5-HT1A-R and βCTF in homogenates of the septal fore-brain. Details of the procedures are reported in Supplementarymethods.

Behavioral testing

Micewere behaviorally tested using theMorrisWaterMaze (MWM)test, the Novel Object Recognition (NOR) test and the Passive Avoidance(PA) test as previously described (Bevins and Besheer, 2006; Gallagheret al., 1993; Loizzo et al., 2013). See Supplementary methods for moredetails.

Statistical analysis

Data from single animals represented the unity of analysis. Resultsare presented as mean ± standard error of the mean (SE). Statisticaltesting was performed using a two-way analysis of variance (ANOVA)

Fig. 1. Experimental protocol. Starting from postnatal day 3 (D3) and up to 15 days (D15) of age, euploid and Ts65Dn pups received one daily injection of either saline or fluoxetine(see Materials and methods). At 2.0–2.5 months of age some animals received one injection of BrdU, in order to evaluate the fate of the surviving cells. Animals were killed one monthlater, at the age of 3.0–3.5 months. Starting from 18 days before sacrifice animals were subjected to the indicated behavioral tests. Abbreviations: BrdU, bromodeoxyuridine; D, day;MWM, Morris Water Maze; NOR, Novel Object Recognition; PA, Passive Avoidance.

206 F. Stagni et al. / Neurobiology of Disease 74 (2015) 204–218

with genotype (euploid and Ts65Dn) and treatment (vehicle, fluoxe-tine) as fixed factors and mouse as random factor or one-way ANOVAfollowed by Fisher LSD post hoc test or Duncan's test. A probabilitylevel of P b 0.05 was considered to be statistically significant.

Results

Effect of neonatal treatment with fluoxetine on the proliferation potency ofneural precursor cells

We estimated with Ki-67 immunohistochemistry the long-termeffect of treatment on the size of the population of actively dividingneural precursor cells (NPCs) in the two major neurogenic niches ofthe postnatal brain, the subgranular zone (SGZ) of the hippocampaldentate gyrus (DG) and the subventricular zone (SVZ) of the lateral ven-tricle. Similar to younger mice (Bianchi et al., 2010), Ts65Dn mice aged3.0–3.5 months had a reduced number of NPCs in comparison with eu-ploid mice both in the DG (Fig. 2A) and SVZ (Fig. 2B). In Ts65Dn mice,

Table 1Immunohistochemistry procedures.

Antigen Application Antibody — dilution

Cleaved caspase-3 IHC Primary: rabbit monoclonal 1:200Secondary: Cy3 conjugated anti-rab

Doublecortin (DCX) IHC Primary: goat polyclonal 1:100Secondary: biotinylated anti-goat IgDAB kit

Ki-67 IHC Primary rabbit monoclonal 1:100Secondary: HRP-conjugated anti-rabDAB kit

Rab5 IHC Primary: mouse monoclonal 1:100Secondary: HRP-conjugated anti-moand DAB kit

Synaptophysin (SYN) IHC Primary: mouse monoclonal 1:1000Secondary: FITC-conjugated anti-mo

5-Bromo-2-deoxyuridine (BrdU) DFIHC Primary: rat monoclonal 1:100Secondary: Cy3 conjugated anti-rat

Glial fibrillary acidic protein (GFAP) DFIHC Primary: mouse monoclonal 1:100Secondary: FITC conjugated anti-mo

Neuronal-specific nuclear protein(NeuN)

DFIHC Primary: mouse monoclonal 1:250Secondary: FITC conjugated anti-mo

IHC, immunohistochemistry; DFIHC, double-fluorescence immunohistochemistry.

which had been neonatally treated with fluoxetine the number ofNPCs became similar to that of untreated euploid mice (Figs. 2A,B). Noeffect of treatment on apoptotic cell death in the DG and SVZ wasobserved either in euploid or Ts65Dn mice (data not shown).

The cyclin-dependent kinase p21 (cip1/WAF1), a cyclin that inhibitscell cycle progression, is over expressed in the trisomic brain(Engidawork et al., 2001; Guidi et al., 2014; Park et al., 2010). Evaluationof p21 levels in hippocampal homogenates showed that adult Ts65Dnmice had high levels of p21 and that neonatal treatmentwith fluoxetinewas followed by a long-term reduction of p21 levels (Fig. 2C,D).

Effect of neonatal treatment with fluoxetine on neuralphenotype acquisition

In order to establish the long-term effect of neonatal treatment onthe differentiation program, we injected mice with BrdU at the age of2.0–2.5 months (i.e. 1.5–2.0 months after treatment cessation) andexamined the phenotype of the surviving cells in the DG after one

Purpose

bit IgG 1:200To label apoptotic cells

G 1:200 andTo label new granule cells and examine their dendritic processes

bit 1:200 andTo label cycling cells (Ki-67 is expressed in all phases of the cell cycleexcept early G1)

use 1:500To label early endosomes

use 1:200To label presynaptic terminals

IgG 1:200To detect BrdU positive cells (surviving cells)

use IgG 1:200To detect surviving cells differentiated into astrocytes

use IgG 1:200To detect surviving cells differentiated into neurons

Fig. 2. Effect of neonatal treatment with fluoxetine on proliferation potency in the DG and SVZ in adulthood. A, B: Total number of Ki-67-positive cells in the DG (A) and SVZ (B) ofuntreated and treated euploid and Ts65Dnmice aged 3.0–3.5months. Values refer to one hemisphere. C, D:Western blot analysis of p21 levels in the hippocampal formation of untreatedand treated euploid and Ts65Dn mice aged 3.0–3.5 months. Western immunoblots in (C) are examples from animals of each experimental group. Histograms in (D) show p21 levelsnormalized to GAPDH and expressed as fold difference in comparison with untreated euploid mice. Values in (A, B, D) represent mean ± SE. *p b 0.05; **p b 0.01 (Duncan's test afterANOVA). Abbreviations: DG, dentate gyrus; SVZ, subventricular zone.

207F. Stagni et al. / Neurobiology of Disease 74 (2015) 204–218

month. In untreated Ts65Dn mice the total number of BrdU positivecells was reduced in comparison with euploid mice (Figs. 3A,B), whichis consistent with their reduced proliferation potency (see Figs. 2A,B).An analysis of the phenotype of the BrdU positive cells showed thatuntreated Ts65Dn mice had, in absolute terms, fewer new neuronsthan untreated euploid mice (Fig. 3C) and, in relative terms, a reducedpercentage of new neurons and an increased percentage of new astro-cytes (Fig. 3D). In Ts65Dn mice neonatally-treated with fluoxetinethere was an increase in the total number of BrdU positive cells (Figs.3A,B), which is consistent with the beneficial impact of treatment onthe proliferation potency (see Fig. 2A). In absolute terms there was anincrease in the number of new neurons and, in relative terms, anincrease in the percentage of new neurons and a reduction in thepercentage of new astrocytes.

Effect of neonatal treatmentwith fluoxetine on total number of granule cells

Doublecortin (DCX) is a marker of immature neurons. In order toestablish the long-term effect of neonatal treatment with fluoxetineon the level of ongoing neurogenesis in adulthood, we evaluated thenumber of DCX positive cells present in the DG. In view of the time-course of DCX expression (about 4 weeks after neuron birth), the oldestDCX positive cells are cells that were born 1.5–2.0 months after treat-ment cessation. While untreated Ts65Dn mice had fewer DCX positivecells in comparison with euploid mice, neonatally-treated Ts65Dnmice had more DCX positive cells (Figs. 4A,B), consistently with thelong-term restoration of the differentiation program (see Figs. 3C,D).Stereological examination of the DG showed that in Ts65Dn mice thegranule cell layer had a smaller volume, reduced cell density andreduced number of granule cells in comparison with euploid mice(Fig. 4C). Consistently with the long-term effect of treatment onneurogenesis, treated Ts65Dn mice showed an increase in the volume

of the granule cell layer, granule cell density and total granule cellnumber (Fig. 4C) that became similar to those of untreated euploidmice.

Effect of neonatal treatment with fluoxetine on granulecell dendritic development

Quantification of the dendritic tree of new granule neurons insections immunostained for DCX showed that Ts65Dn mice had ashorter dendritic length, and a reduced number of segments (Fig. 5C),longer branches of orders 1–3 and 7 (Fig. 5D, upper panel) and fewerbranches of orders higher than 3 (Fig. 5D, lower panel) in comparisonwith euploid mice. All these defects were completely rescued by treat-ment (Figs. 5C,D). Consequently, as shown by the dendrogram inFig. 5B, treated Ts65Dn mice had a dendritic pattern that was similarto that of untreated euploid mice. Since DCX is expressed from one tofour weeks after neuron birth, the normal dendritic pattern of granuleneurons in treated Ts65Dn mice aged 3.0–3.5 months implies thatneonatal treatment with fluoxetine positively impacts dendritic devel-opment of neurons that were born 1.5–2.0 months after treatmentcessation.

In order to establish the long-term effects of treatment on thedendritic tree of old granule cells, we examined Golgi-stained granuleneurons located in the outer portion of the granule cell layer, the zonethat harbors the oldest granule cells (Amaral and Witter, 1995).Ts65Dn mice had a shorter dendritic length and a reduced number ofsegments (Fig. 5G), abnormally long branches of orders 1 and 2(Fig. 5H, upper panel), fewer branches of orders 3–5 and a lack ofbranches of a higher order (Fig. 5H, lower panel) compared to euploidmice. All these defects were completely rescued by treatment (Figs.5G,H). Consequently, as shown by the dendrogram (Fig. 5F), treated

Fig. 3. Effect of neonatal treatmentwith fluoxetine on the phenotype of cells born in the dentate gyrus in adulthood. A, B: Examples of sections processed for fluorescent immunostainingfor BrdU (the arrow shows a BrdU positive cells) in the DG of an animal of each group (A) and total number of BrdU positive cells (B) in untreated and treated euploid and Ts65Dnmiceaged 3.0–3.5months. Values refer to one hemisphere. These animals were injectedwith BrdU at twomonths of age. Calibration: 100 μm. C, D: Absolute number (C) and percentage (D) ofsurviving cells with a neuronal phenotype (NeuN/BrdU), an astrocytic phenotype (GFAP/BrdU) and an undetermined phenotype (Neither/BrdU) in the DG (granule cell layer + hilus) ofuntreated and treated euploid and Ts65Dnmice. Same animals as in (B). Values in (B, C) represent totals for one DG. Values in (B, C, D) are mean ± SE. *p b 0.05; **p b 0.01; ***p b 0.001(Duncan's test after ANOVA). Abbreviations: Gr, granule cell layer; SGZ, subgranular zone.

208 F. Stagni et al. / Neurobiology of Disease 74 (2015) 204–218

Ts65Dnmice had a dendritic pattern thatwas similar to that of untreatedeuploid mice.

Effect of neonatal treatment with fluoxetine on hippocampal synapses

Ts65Dn mice had a reduced spine density in the inner and outerparts of the dendritic tree of the granule cells (Fig. 6A) and in the prox-imal dendritic tree of CA3 pyramidal neurons (Fig. 6B). In treatedTs65Dn mice these defects were fully rescued (Figs. 6A,B).

We examined the immunoreactivity for synaptophysin (SYN), aspecific marker of presynaptic terminals, in the molecular layer of theDG and in the stratum lucidum of field CA3, the site of termination ofthe axons of the granule cells. In untreated Ts65Dnmice the optical den-sity (OD) of SYNwas significantly lower than in untreated euploidmiceboth in the DG (Figs. 6C,E) and field CA3 (Figs. 6D,F). In Ts65Dn miceneonatally-treated with fluoxetine there was an increase in the OD ofSYN both in the DG (Figs. 6C,E) and CA3 (Figs. 6D,F). We evaluated thedensity of individual puncta exhibiting SYN immunoreactivity in orderto establish if the cause of the observed differences in immunoreactivitywas a change in the number of synaptic terminals. While untreatedTs65Dn mice had fewer puncta exhibiting SYN immunoreactivity bothin the DG (Figs. 6G,I) and CA3 (Figs. 6H,J), in neonatally-treated

Ts65Dnmice the density of SYN puncta was similar to that of untreatedeuploid mice (Figs. 6G,I,H,J).

Effect of neonatal treatment with fluoxetine on behavioral tasks

Animals were subjected to hippocampus-dependent behavioraltasks (Crawley, 2007) before sacrifice. We used the Morris WaterMaze (MWM), the Novel Object Recognition (NOR) and the PassiveAvoidance (PA) tests. The latter test is also amygdala-dependent(Babri et al., 2007; Riekkinen et al., 1993). Themajor results are reportedbelow (for a more detailed description see Supplementary results).

Ts65Dn (untreated: n=17, treated: n=12) and euploid (untreated:n = 20, treated: n = 18) mice were tested for their ability to find ahidden platform in the MWM for 5 days and were subjected to theprobe test on day 6 (Fig. 7A). The following parameterswere consideredas an index of spatial memory: i) latency to enter the former platformquadrant, ii) percentage of time spent in the former platform quadrant,iii) frequency of entrances in the former platform quadrant, and iv)proximity to the former platform position (Gallagher's test). Perfor-mance in all examined parameters was severely impaired in untreatedTs65Dn mice. They showed an increase in the latency to enter theformer platform quadrant (Fig. 7B), a reduction in the time spentthere (Fig. 7C) and in the frequency of entrances (Fig. 7D), and a

Fig. 4. Effect of neonatal treatment with fluoxetine on granule cell number in adulthood. A: Hippocampal sections immunostained for DCX from animals of each experimental group. DCXpositive cells were localized close to the inner border of the granule cell layer, in agreement with the location of neural precursor cells of the DG. Calibration: 100 μm. B: Number (mean±SE) of DCX positive cells over a length of 100 μm in each experimental group. C: Volume of the granule cell layer (left panel), density of granule cells (middle panel) and total number ofgranule cells (right panel) in untreated and treated euploid and Ts65Dnmice aged 3.0–3.5 months. Values (mean ± SE) refer to one DG. *p b 0.05; **p b 0.01; ***p b 0.001 (Duncan's testafter ANOVA). Abbreviations: Gr, granule cell layer; H, hilus; Mol, molecular layer.

209F. Stagni et al. / Neurobiology of Disease 74 (2015) 204–218

reduction in proximity to the platform quadrant (Fig. 7E). Remarkably,in neonatally-treated Ts65Dn mice all these parameters were fullyrestored (Figs. 7A–E).

Ts65Dn mice (untreated: n = 14, treated: n = 9) and euploid mice(untreated: n = 9, treated: n = 12) were tested with NOR. This test isbased on the fact that mice preferentially explore a novel object whenpresented with a pair of objects consisting of an already explored anda novel object. Therefore, an increased exploration of the novel objectmeans that the animal remembers that the old object had alreadybeen explored. Untreated Ts65Dn mice explored the new object lessthan did euploid mice 1 h after the initial presentation of the objects,i.e. they had a reduced discrimination index (Fig. 7F). Importantly, inneonatally-treated Ts65Dnmice the discrimination index became simi-lar to that of euploidmice (Fig. 7F), indicating restoration of recognitionmemory. No differences among groups were observed after 24 h(Fig. 7G), indicating no effect of genotype and treatment on the long-term retention aspects of this task.

In the PA test an animal is conditioned with a single aversive eventand is later tested for recollection of that experience. Figs. 7H,I report

the latency time to enter the dark compartment on the first day (train-ing) and on the second day (test) of the behavioral procedure forTs65Dn (untreated: n = 16, treated: n = 12) and euploid (untreated:n = 20, treated: n = 19) mice. On the first day, all groups showedsimilar step-through latencies (Fig. 7H). After 24 h (second day),animals were re-placed in the test apparatus to test their memory.Untreated Ts65Dn mice were severely impaired in this task as shownby a reduced latency to enter the dark compartment in comparisonwith euploid mice (Fig. 7I). In neonatally-treated Ts65Dn mice thelatency underwent an increase and was no different in comparisonwith that of euploid mice (Fig. 7I).

Effect of neonatal treatment with fluoxetine on the expression of theserotonin receptor 5-HT1A and plasticity-related molecules

The 5HT1A-R, which plays a key role in neurogenesis and synapto-genesis, exhibits a reduced expression in the trisomic brain (Bar-Peledet al., 1991; Bianchi et al., 2010; Guidi et al., 2013). We examined the5-HT1A-R in the hippocampus in order to establish whether neonatal

Fig. 5. Effect of neonatal treatment with fluoxetine on the dendritic architecture of granule cells. A–H: dendritic architecture of new (A–D) and older (E–H) granule cells of treated anduntreated euploid and Ts65Dn mice aged 3.0–3.5 months. A, E: Examples of the dendritic tree of granule cells (g1–g4) from animals of each experimental group (g1: euploid; g2:Ts65Dn; g3: treated euploid; g4: treated Ts65Dn mice). New granule cells (A) were immunostained with DCX and older granule cells (E) were Golgi-stained. Numbers indicate the dif-ferent dendritic orders. Calibration: 10 μm (A) and 20 μm (F). B, F: Dendrograms of new (B) and older (F) granule cells, obtained from the mean length and mean number of branchesof each order reported in (D) and (H), respectively. The number of branches was approximated to the nearest integer value (thick lines). Thin lines have been used to indicate a numberof branches ranging from 0.1 to 0.5. Calibration: 10 μm (B) and 50 μm (F). C, G: Total dendritic length (upper panel) and total number of branches (lower panel) of new (C) and older(G) granule cells D, H: Mean length (upper panel) and mean number (lower panel) of branches of the different orders of new (D) and older (H) granule cells. Arrows in (H) indicatelack of branches of orders 6 and 7 in untreated Ts65Dn mice. Values in (C, D, G, H) represent mean ± SE. *p b 0.05; **p b 0.01; ***p b 0.001 (Duncan's test after ANOVA).

210 F. Stagni et al. / Neurobiology of Disease 74 (2015) 204–218

Fig. 6. Effect of neonatal treatment with fluoxetine on hippocampal synapses in adulthood. A, B: Spine density in the proximal and distal dendritic branches of the granule cells (A) and inthe dendritic shaft of CA3 pyramidal neurons (B) of untreated and treated euploid and Ts65Dnmice aged 3.0–3.5 months. C, D: Images of sections processed for synaptophysin immuno-fluorescence from the DG (C) and field CA3 (D) from an animal of each experimental group. Calibration: 50 μm. E, F: Optical density of synaptophysin immunoreactivity in the inner,middle and outer third of the molecular layer of the DG (E) and the stratum lucidum of CA3 (F) of untreated and treated euploid and Ts65Dn mice aged 3.0–3.5 months. Data aregiven as fold difference vs. inner molecular layer (E) and stratum lucidum (F) of untreated euploid mice. G, H: Images taken with the confocal microscope from the middle molecularlayer (G) and stratum lucidum of CA3 (H). Calibration: 3 μm. I, J: Number of puncta per μm2 exhibiting synaptophysin immunoreactivity in the molecular layer of the DG (I) and thestratum lucidum of field CA3 (J) of untreated and treated euploid and Ts65Dnmice aged 3.0 3.5 months. Values in (A, B, E, F, I, J) represent mean± SE. **p b 0.01; ***p b 0.001 (Duncan'stest after ANOVA) . Abbreviations: Gr, granule cell layer; i, inner; m, middle; L, stratum lucidum; Mol, molecular layer; o, outer; Pyr, pyramidal layer.

211F. Stagni et al. / Neurobiology of Disease 74 (2015) 204–218

treatment had a long-term effect on its expression. We additionallyexamined the phosphorylation (activity) of Erk1/Erk2 proteins thatare the key effectors of serotonergic signaling (Cowen, 2007). UntreatedTs65Dn mice had reduced levels of the 5-HT1A-R in comparison with

euploid mice (Figs. 8A,B), which may explain the persistence ofneurogenesis and neuron maturation defects at adult life stages. Erk1/Erk2, which are triggered by activation of various serotonin receptors,including the 5-HT1A-R (Cowen, 2007) are involved in various plasticity

Fig. 7. Effect of neonatal treatment with fluoxetine of cognitive performance in adulthood. A–E: Spatial learning assessed with the Morris Water Maze. Ts65Dnmice that received saline(empty squares) did not learn over the 5-day period. Euploid mice that received either saline (open circles) or fluoxetine (filled circles) did learn with an approximate halving of theirlatency. Ts65Dn mice that received fluoxetine (filled squares) learned over the 5-day period better than their untreated counterparts (A). On day six (probe test) Ts65Dn mice stillfound the platformwith a longer latency in comparison with euploid mice (B), spent a reduced amount of time in the quadrant of the platform (C), entered the quadrant with a reducedfrequency (D) and swam at a longer distance from the platform (E) in comparison with euploid mice. In treated Ts65Dnmice the latency (B), percentage of time spent in the quadrant oftheplatform(C), the frequency of entrances (D) and theproximity to theplatform (E) became similar to those of euploidmice. F, G:NovelObject Recognition test. The discrimination indexfor the object recognition test showed a deficiency in Ts65Dnmice after a retention period of 1 h. This defect was rescued by treatment (F). After a retention period of 24 h no differenceswere found among groups (G). H, I: Passive Avoidance test. Graphs show the latency time for entering the dark compartment on the first day (H) and on the second day (I) of the behav-ioral procedure. On the first day, mice were conditioned with an electrical foot shock when they entered the dark compartment (conditioning). At 24 h after the foot shock, mice werereintroduced into the light–dark box and the time it took mice to enter the dark compartment (light–dark latency) was measured. Values represent mean ± SE. (*) p b 0.06; *p b 0.05;**p b 0.01; ***p b 0.001 (Fisher LSD test after ANOVA). Abbreviations: Tn, exploration time of novel object; To, exploration time of old object.

212 F. Stagni et al. / Neurobiology of Disease 74 (2015) 204–218

cascades (Thomas and Huganir, 2004). Consistent with previousevidence (Altafaj et al., 2013), Ts65Dn mice exhibited a reducedpErk1/Erk1 ratio (Figs. 8B,C). Here, we additionally found that Ts65Dnmice also exhibited a reduced pErk2/Erk2 ratio (Figs. 8B,C), indicatinga generalized impairment of Erk1/Erk2 signaling. Importantly, inneonatally-treated Ts65Dnmice the levels of the 5-HT1A-Rwere similarto those euploid mice (Figs. 8A,B) and this effect was accompanied byrestoration of Erk1/Erk2 activity (Figs. 8C,D), indicating a long-termimpact of treatment on 5-HT1A-R expression and 5-HT1A-R-mediated

signaling. The expression of the methyl-CpG-binding protein (MeCP2),the mutations of which cause the neurodevelopmental disorder Rett'ssyndrome (Amir et al., 1999) has been shown to be down-regulated inDS (Kuhn et al., 2010). MeCP2 activates the expression of numerousgenes, including the 5-HT1A-R (Chahrour et al., 2008), making MeCP2a good candidate for the 5-HT1A-R reduced expression in the trisomicbrain. We found that the reduced levels of MeCP2 in Ts65Dn miceunderwent an increase after neonatal treatment with fluoxetine andbecame similar to those of euploid mice (Figs. 8E,F). This may provide

Fig. 8.Effect of neonatal treatmentwith fluoxetine on the hippocampal expression of the serotonin receptor 5-HT1A andplasticity-relatedmolecules in adulthood.Western blot analysis ofthe 5-HT1A-R, Erk1/Erk2, MeCP2 and BDNF in hippocampal homogenates of untreated and treated euploid and Ts65Dn mice aged 3.0–3.5 months. A, B: Representative blots (A) andcumulative data (B) for the 5-HT1A-R. C, D: Representative blots (C) and cumulative data (D) for pErk1/2 and total Erk1/2. E, F: Representative blots (E) and cumulative data (F) forMeCP2. G, H: Representative blots (G) and cumulative data (H) for BDNF. Levels of the 5-HT1A-R, Erk1/Erk2, MeCP2 and BDNF were normalized to GAPDH. Levels of pErk1 and pErk2were normalized to total Erk1 and Erk2, respectively. Data in (B, D, F, H) (mean ± SE) are expressed as fold difference in comparison with untreated euploid mice. *p b 0.05;**p b 0.01; ***p b 0.001 (Duncan's test after ANOVA).

213F. Stagni et al. / Neurobiology of Disease 74 (2015) 204–218

a mechanistic link with the observed normalization of 5-HT1A-Rexpression and restoration of MeCP2 protein levels. It has been shownthat treatmentwith fluoxetine increases brain MeCP2 levels most likelythrough serotonergic signaling (Cassel et al., 2006). Future work isneeded to elucidate the mechanisms whereby fluoxetine increasesMeCP2 expression in Ts65Dn mice. Brain-derived neurotrophic factor(BDNF), a protein that plays a key role in neurogenesis and neuronmaturation, is down-regulated in the brain of neonate Ts65Dn mice(Bianchi et al., 2010). Since MeCP2 directly transcribes BDNF, we won-deredwhether normalization ofMeCP2 levels also had a positive impacton BDNF expression in Ts65Dn mice treated with fluoxetine. We found

that in Ts65Dnmice BDNF levels, similar to those of the 5-HT1A-R, werefully reinstated by treatment (Figs. 8G,H).

Effect of neonatal treatment with fluoxetine on APP processing

Early endosomal alterations are the earliest pathology in AD and DS(Cataldo et al., 2003) and may be one of the mechanisms underlyingneurodegenerative changes during the progression of AD. AD-relatedendosome dysfunction in DS is dependent on elevated levels of βCTF(Jiang et al., 2009). Up-regulation of Rab5, a GTPase of early endosomes,has been detected in AD and DS brain samples and is correlated with

214 F. Stagni et al. / Neurobiology of Disease 74 (2015) 204–218

cognitive decline (Cataldo et al., 2003; Ginsberg et al., 2011). Moreover,Rab5 is markedly up-regulated in DS fibroblasts, and altering Rab5expression creates a similar endosomal phenotype as that resultingfrom the manipulation of βCTF (Cataldo et al., 2008). The septal area isa brain region where Ts65Dn mice show endosome alterations startingfrom 2 months of age (Cataldo et al., 2003). In a mouse model of

Fig. 9.Effect of neonatal treatmentwithfluoxetineon early endosomes andAPP processing in adsampled within the area enclosed in the dotted box. The higher magnification image at the toimage); 20 μm (high magnification image). B, C: Examples of Rab5 immunopositive cells fromof the septal region (C) of untreated and treated euploid and Ts65Dnmice aged 3.0–3.5 monthenates containing the septal region of untreated and treated euploid and Ts65Dn mice aged 3.0homogenates of untreated and treated euploid and Ts65Dnmice aged3.0–3.5months. RepresenBACE1were normalized to GAPDH. Data (mean±SE) in (C, E) are expressed as fold difference inafter ANOVA). Abbreviations: AC, anterior commissure; CC, corpus callosum; SR, septal region;

Alzheimer's disease (AD) serotonergic signaling appears to modifyAPP processing through the 5-HT1A-R, with a reduction in the produc-tion of the β-amyloid peptide (Cirrito et al., 2011; Nelson et al., 2007).Thus, we wondered whether treatment with fluoxetine in Ts65Dnmice has an effect on APP processing, thereby preventing endosomeabnormalities. Evaluation of Rab5 levels in neurons of the septal area

ulthood. A: Coronal brain section encroachingon the septal region. Rab5 positive cellswerep shows examples of Rab5 positive cells (arrows). Calibrations: 1 mm (low magnificationthe septal region of an animal of each group (B) and expression levels of Rab5 in neuronss. Calibration: 5 μm. D, E: Expression levels of βCTF (D) and 5-HT1A-R (E) in brain homog-–3.5 months. F–H: Expression levels of APP (F), BACE1 (G) and βCTF (H) in hippocampaltativeblots are reported below thehistograms in (D–H). Levels ofβCTF, 5-HT1A-R, APP andcomparisonwith untreated euploidmice. *p b 0.05; **p b 0.01; ***p b 0.001 (Duncan's testSTR, striatum.

215F. Stagni et al. / Neurobiology of Disease 74 (2015) 204–218

showed that, while Rab5 was up-regulated in untreated Ts65Dn mice,after neonatal treatment with fluoxetine Rab5 levels underwent areduction and became similar to those of untreated euploid mice(Fig. 9C). Homogenates from the septal forebrain exhibited increasedβCTF levels (Fig. 9D) and reduced levels of the 5-HT1A-R (Fig. 9E) inuntreated Ts65Dn mice. These defects were fully rescued by neonataltreatment with fluoxetine (Figs. 9D,E).

Previous evidence in Ts65Dn mice showed no endosome abnormal-ities in the hippocampal formation before the age of 12months (Cataldoet al., 2003). Accordingly, we found very low Rab5 immunoreactivity inthe hippocampus of Ts65Dnmice (data not shown). Since accumulationof APP cleavage products may precede endosome alterations, we exam-ined products of the amyloidogenic pathway in untreated mice and inmice which had been neonatally-treated with fluoxetine. We foundthat Ts65Dn mice had higher levels of APP that were not affected bytreatment with fluoxetine (Fig. 9F). Increased levels of BACE1, theenzyme that cleaves APP at the β-site, thereby producing βCTF (Caiet al., 2001), are a typical feature of DS (Sun et al., 2006). We examinedthe levels of BACE1 and found that the hippocampus of untreatedTs65Dn mice had higher BACE1 levels than that of their euploid coun-terparts (Fig. 9G). Importantly, in neonatally-treated Ts65Dn miceBACE1 levels underwent a reduction and became similar (Fig. 9G) tothose of euploid mice. We next examined the hippocampal levels ofβCTF and found that, while untreated Ts65Dn mice had high βCTFlevels, in neonatally-treated Ts65Dn mice βCTF levels were fullynormalized and became similar to those of euploid mice (Fig. 9H).

Discussion

Results show that a brief neonatal treatmentwith fluoxetine leaves along-term positive trace in the brain of Ts65Dnmice so that whenmicereach adulthood the hippocampus and hippocampus-dependent cogni-tive performance are similar to those of control mice and no early signsof AD-like pathology are present.

A brief neonatal treatment with fluoxetine induces long-term restoration ofthe hippocampal architecture and cognitive performance in Ts65Dn mice

The majority of granule neurons are produced in the first two post-natal weeks (Altman and Bayer, 1990; Schlessinger et al., 1975) andthe process of dendritic maturation lasts 50–60 days (Rhin andClaiborne, 1990; Zhao et al., 2006). This implies that the first postnatalperiod, when most of the new granule neurons are produced and theirdendrites start to mature and receive synaptic contacts, is a particularlycritical time window. Previous evidence has shown recovery ofneurogenesis, dendritic size, spine density, connectivity, volume andcellularity in the hippocampal DG of adolescent Ts65Dn miceneonatally-treated with fluoxetine (Bianchi et al., 2010; Guidi et al.,2013; Stagni et al., 2013). Current results show that adult Ts65Dnmice neonatally-treated with fluoxetine still exhibit fully normalizedneurogenesis in the DG and SVZ. Embryos and infants with DS(Engidawork et al., 2001; Park et al., 2010) and adolescent Ts65Dnmice (Guidi et al., 2014) exhibit high brain levels of p21 (cip1/WAF1),a cyclin-dependent kinase that inhibits cell cycle progression, suggest-ing that excessive p21 levels may be a determinant of neurogenesisimpairment in DS. In euploid mice, inhibition of p21, associated withan increase in hippocampal neurogenesis, was previously described24 h after chronic treatment with fluoxetine (Pechnick et al., 2011).We show here that neonatal treatment with fluoxetine is followed bylong-term normalization of p21 expression in adult Ts65Dn mice. Thiseffect may explain the long-lasting normalization of NPC proliferationpotency. A more recent study (Huang et al., 2012) highlights a verycomplex molecular signature of fluoxetine treatment in the brain,with changes in specific networks of genes, suggesting that additionalgenes/pathways may be involved in the long-term restoration ofneurogenesis.

An altered differentiation programcharacterizes trisomic NPCs, witha reduction in the number of cells that acquire a neuronal phenotypeand a relative increase in the number of cells that acquire an astrocyticphenotype (Contestabile et al., 2009; Contestabile et al., 2007; Guidiet al., 2008). Current results show restoration of phenotype acquisitionin treated Ts65Dn mice indicating a treatment-induced long-termcorrection of the aberrant differentiation program that characterizestrisomic NPCs. In neonatally-treated Ts65Dnmice the volume and cellu-larity of the DG were similar to those of euploid mice, indicating thatneonatal treatment with fluoxetine has a long-term impact on the pro-duction of granule cells that spans to adulthood.While a previous studyin transgenicmicewith reduced BDNF levels or impaired trkB activationshowed an increase in apoptotic cell death shortly after chronic treat-ment with fluoxetine (Sairanen et al., 2005), we found here that inadult Ts65Dn mice that had been neonatally-treated with fluoxetinethere was no increase in cell death. This indicates that long-term resto-ration of neurogenesis of granule cells is not counteracted by a cell deathincrease.

Dendritic hypotrophy and spinedensity reduction are typical defectsof DS. Results show that adult Ts65Dn mice neonatally-treated withfluoxetine had granule cells with normal dendritic tree and normalspine density, indicating that the restoration of dendritic architectureobserved in adolescent Ts65Dn mice (Guidi et al., 2013) is still presentin adulthood. In parallel with restoration of dendritic development,treated Ts65Dnmice exhibited normalization of hippocampal synapsesin both theDG andfield CA3. This evidence shows that the restoration ofconnectivity observed in the DG and CA3 of adolescent Ts65Dn miceneonatally-treated with fluoxetine (Guidi et al., 2013; Stagni et al.,2013) is still present at 3.0–3.5months of age, indicating that the rescueof connectivity is durably retained.

Taken together our results show that, not only does a neonatal treat-ment lead to a reversal of the trisomy-linked neurodevelopmentaldisorders, but that this effect is enduringly retained. The long-termrestoration of hippocampal cellularity, dendritic architecture and con-nectivity may not necessarily lead to a positive impact on hippocampalfunction. For this reasonwe examined cognitive performance and foundthat the functional correlate of these effects was restoration of behavior.Thus, the long-term impact of neonatal treatment with fluoxetine onthe hippocampus of Ts65Dn mice translates into a long-term rescue ofmemory functions.

Neonatal treatment with fluoxetine enduringly restores the expression ofthe serotonin receptor 5-HT1A

The results discussed above show that an early and brief treatmentwith fluoxetine has a rescue effect that spans to adulthood. It must benoted that various trisomic genes such as APP, DYRK1A, and DSCAMare likely to be involved in brain alterations in DS and it is probablethat these genes are still over expressed in treated Ts65Dn mice. Thecurrent finding that APP is still over expressed in neonatally-treatedadult Ts65Dn mice supports this idea. The question thus arises of howa short treatment with fluoxetine in the neonatal period is sufficientto induce effects that last to adulthood.

The trisomic brain is characterized by reduced expression of the5-HT1A receptor starting from early life stages (Bar-Peled et al., 1991;Bianchi et al., 2010; Guidi et al., 2014). Since serotonin plays a pivotalrole in neurogenesis and dendritic development (Whitaker-Azmitia,2001) and it requires the 5-HT1A-R for this (Santarelli et al., 2003;Yan et al., 1997a, 1997b), the reduced expression of the 5-HT1A-Rduring early life stages is most likely one of the determinants of theimpairment of brain development in DS. Consistent with this idea,previous results (Bianchi et al., 2010) showed that neonatal treatmentwith fluoxetine reinstates the 5-HT1A-R expression that is paralleledby restoration of neurogenesis. There is evidence that chronic treatmentwith fluoxetine causes desensitization of the 5-HT1A autoreceptors inthe raphe area, thereby increasing serotonin signaling (Le Poul et al.,

216 F. Stagni et al. / Neurobiology of Disease 74 (2015) 204–218

2000). Therefore, desensitization of the serotonin 5-HT1A autoreceptorsmay additionally contribute to the short-term effects of treatment onneurogenesis.

Current results show that the effects of fluoxetine outlive cessationof treatment by 2.5–3.0 months. After drug washout, desensitizationof serotonin autoreceptors, causing changes in serotonin signaling,rapidly disappears (Anthony et al., 2000). Thus, it is unlikely that thismechanism plays a role in the long-lasting effects of treatment. Wefound here that the expression of the 5-HT1A-R was still normalizedwhen neonatally-treated Ts65Dn mice reached adulthood. This long-term effect may explain why the brain and behavioral phenotype ofTs65Dn mice remained restored well after treatment cessation.

An intriguing issue dealswith themechanism/s underlying the long-term normalization of the expression of the 5-HT1A-R. Consistent withprevious evidence in individuals with DS (Kuhn et al., 2010) we foundhere that Ts65Dn mice had reduced MeCP2 levels. In view of the roleplayed by this gene in the regulation of the 5-HT1A-R (Chahrour et al.,2008), the long-term restoration of MeCP2 protein levels induced bytreatment in Ts65Dn mice may explain the long-term normalization ofthe 5-HT1A-R. Since it has been shown that serotonin signalingpositively affects MeCP2 expression (Cassel et al., 2006), restoration ofserotonin signaling, due to restoration of the 5-HT1A-R expression,may exert, in turn, a positive feedback on MeCP2 expression. In agree-ment with restoration of MeCP2 levels in treated Ts65Dn mice, wealso found that BDNF levels were normalized. The latter effect maycontribute to the long-term positive impact of neonatal treatmentwith fluoxetine on the trisomic brain. Though current results suggestthat the long-term restoration of serotonin signaling may underlie thelong-term effects of fluoxetine, it cannot be ruled out that other signal-ing pathways are involved in the long-term rescue of the brain andbehavioral phenotype of Ts65Dn mice.

Neonatal treatment with fluoxetine impacts APP processing

APP is one of the triplicated genes thought to contribute to develop-ment of AD in individuals with DS. We found here increased APP levelsin Ts65Dn compared to euploid mice. This is in agreement with studiesshowing increased APP expression both at the RNA and protein levels inthe brain of Ts65Dnmice, starting from very early life stages (Lyle et al.,2004; Netzer et al., 2010; Sultan et al., 2007). Surprisingly, other studiesfailed to find significant differences in APP levels in mice younger than12 months (Choi et al., 2009; Hunter et al., 2003a, 2003b; Seo andIsacson, 2005). It must be observed, however, that from the illustrationsin the articles by Hunter et al. and Seo and Isacson, it appears that, inTs65Dn mice younger than 12 months, APP levels were higher in com-parison with those of the euploid counterparts, though the differencewas not significant. Considering that the difference between Ts65Dnand euploid mice in APP levels is relatively small at young ages andundergoes a large increase with age (Hunter et al., 2003a, 2003b), it ispossible that significant differences between young Ts65Dn and euploidmice are missed due to a high variance.

We found that in neonatally-treated Ts65Dn mice APP levels werelarger than in euploid mice, indicating that fluoxetine does not affectAPP expression. Increased levels of BACE1, the most important brainβ-secretase, are a typical feature of DS (Sun et al., 2006) and in tandemwith triplication of APP cause excessive amounts of βCTF, the source ofβ-amyloid. We found that Ts65Dn mice had increased levels of BACE1,similar to those of humans. Importantly, in adult Ts65Dn mice whichhad been neonatally treated with fluoxetine there was a reduction inthe expression of BACE1 that became fully normalized. A recent studyshows that inhibition of GSK3β activity reduces BACE1 expression (Lyet al., 2013). Regarding this link, it seems of relevance to observe thatTs65Dn mice exhibit excessive activity of GSK3β and that treatmentwith fluoxetine normalizes GSK3β activity through the serotonin5-HT1A-R (Trazzi et al., 2014). Based on this evidence it seems plausible

that the normalization of BACE1 levels found here in neonatally-treatedTs65Dn mice may be due to a reinstatement of GSK3β activity.

Correlatingwith the reduction of BACE1 levels in Ts65Dnmice, βCTFlevels also became normalized. Accumulation of βCTF is the cause ofearly endosomeabnormalities (Jiang et al., 2009) that start to bepresentin DS and AD before themanifest impairment of cognitive functions.Wefound that, consistently with the normalization of βCTF levels, in theseptal region of neonatally-treated Ts65Dnmice early endosome abnor-malities were corrected. In view of the role of serotonin signaling in APPprocessing (Cirrito et al., 2011), the finding that the expression of the5-HT1A-R was restored in neonatally-treated Ts65Dn mice suggeststhat restoration of serotonergic signaling may be involved in theobserved restoration of APP processing and endosomealterations. How-ever, additional factors/pathways may contribute to explain the effectsof treatment on APP processing and endosome abnormalities. Forinstance, treatment may affect the expression levels of Synaptojanin1(SYNJ1), a gene critically involved in derangement of the endocyticpathway in DS lymphoblastoid cell lines (Cossec et al., 2012). Furtherinvestigations are needed in order to clarify the molecular pathwayunderlying the long-term effects of fluoxetine on APP processing.

Taken together, the results show that, not only does early treatmentwith fluoxetine restore brain development and cognitive functions, butalso normalizes the aberrantly high beta-amyloidogenic processing ofAPP in Ts65Dn mice, thereby counteracting the increased expressionof APP. Interestingly, a brain positron emission tomography study ofnormal humans shows a decreased Aβ plaque burden in participantswho had been taking selective serotonin reuptake inhibitors, includingfluoxetine (Cirrito et al., 2011). Ts65Dn mice start to exhibit cognitivedecline at about six months of age (Hunter et al., 2003a, 2003b). Infuture studies we plan to establish whether fluoxetine given at laterlife stages, but before the age of cognitive decline, is still able to preventAD-like pathology. Our results suggest that early treatment with fluox-etine in children/adolescents with DSmight represent a potential strat-egy to prevent the occurrence of AD-like pathology in adulthood. Itmust be observed that the doses of fluoxetine used in clinical trials inchildren (DeLong et al., 2002; Hollander et al., 2005) are lower thanthose used in the current study in mice that correspond to doses usedin adults (Caccia et al. et al., 1990; DeVane, 1994). Therefore, it remainsto be established whether a lower dose of fluoxetine at early life stagesis equally effective in reinstating brain development and preventingAD-like pathology.

Neonatal treatment with fluoxetine has a moderate long-termimpact in euploid mice

In contrast with the impressive effects of neonatal treatment withfluoxetine in Ts65Dn mice, in euploid mice treatment had much moremoderate effects. Though there was an increase in neurogenesis(Figs. 2, 3), spine density, number of dendritic branches and of synapticterminals (Figs. 5, 6), these effects did not lead to behavioral improve-ment (Fig. 7). It seems likely that in normal mice the organization ofbrain circuits is already optimized and, therefore, no further benefitcan be obtained by increasing neurogenesis and connectivity.

Pharmacotherapies in the trisomic brain: timing is all

Various therapies have been attempted in DSmousemodels at adultlife stages, with the goal to improve hippocampus-dependent learningand memory (Contestabile et al., 2013; Fernandez et al., 2007;Lockrow et al., 2010; Salehi et al., 2009). Taken together, results ofpharmacotherapies at adult life stages suggest that an improvement inhippocampal structure and function can be achieved, though theseeffects appear to be in some cases moderate and ephemeral. This isnot surprising, since interventions during adulthood are unlikely tofully restore trisomy-linked brain abnormalities for two major reasons.First, neurogenesis is a prenatal or early postnatal event, in the case of

217F. Stagni et al. / Neurobiology of Disease 74 (2015) 204–218

the hippocampal dentate gyrus. Thus, brain and hippocampalhypocellularity cannot be restored in adulthood. Second, though thedendritic arbor of the neurons forming the brain exhibits a degree ofplasticity, a full rearrangement of the dendritic arbor in adulthood isunlikely. Thus, after the period of neuron proliferation and maturation,relatively little can be done to increase the quantity of neurons formingthe brain and rescue their connections. Our previous studies showedthat a neonatal pharmacotherapy with fluoxetine is able to fully rescuehippocampal development (Bianchi et al., 2010; Guidi et al., 2013;Stagni et al., 2013). The current study shows that during the time inter-vening between treatment cessation (at two weeks of age) and attain-ment of adulthood none of the short-term effects of treatmentdisappeared, indicating that reinstatement of the hippocampal pheno-type and memory performance are stably retained. To our knowledge,this is the first demonstration that a short pharmacotherapy duringthe most critical time window for hippocampal development issufficient to enduringly reverse the trisomy-linked hippocampalneurodevelopmental defects and restore memory. We previouslyfound that prenatal therapywithfluoxetine restores overall brain devel-opment in Ts65Dnmice (Guidi et al., 2014). In future studies we plan toestablish whether such a generalized restoring effect is retained inadulthood. If the effects of a prenatal therapy, similar to those of a neo-natal therapy, endure to adulthood, prenatal treatments may representan extraordinary means for the permanent reversal of the abnormalbrain phenotype in DS.

The Ts65Dn mouse is considered a good model of DS because itexhibits several abnormal phenotypes that are similar to those seen inhuman trisomy 21. It must be observed, however, that this, as well asother DS mouse models, poses unavoidable limitations because it istrisomic for different sets of genes which are orthologous to those ofHsa21 (Liu et al., 2011). Caution, therefore, must be exercised in thetranslation of results from animal models to the human disorderbecause the outcome may be different. Antidepressants during preg-nancy and during early life stages may have adverse effects, such asgrowth restriction and a neonatal withdrawal syndrome, though theseeffects seem to disappear with time (Einarson et al., 2009; Hayes et al.,2012; Olivier et al., 2013; Pedersen et al. et al., 2010). Yet, even if theuse of fluoxetine may pose some caveats regarding translation tohumans, our studies provide proof-of-concept evidence that the DSphenotypemay be enduringly corrected with early pharmacotherapies.In the future, we plan to examine the effect of additional drugs on thetrisomic brain, in order to identify those that have a positive impactsimilar to that of fluoxetine but no (or scarce) side effects.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgment

This work was supported by grants to R. B. from the “FondationJerome Lejeune” and University of Bologna (RFO2012BART;RFO2013BART). The technical assistance of Mr. Francesco Campisi isgratefully acknowledged.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.nbd.2014.12.005.

References

Altafaj, X., Martin, E.D., Ortiz-Abalia, J., Valderrama, A., Lao-Peregrin, C., Dierssen, M., Fillat, C.,2013. Normalization of Dyrk1A expression by AAV2/1-shDyrk1A attenuateshippocampal-dependent defects in the Ts65Dn mouse model of Down syndrome.Neurobiol. Dis. 52, 117–127.

Altman, J., Bayer, S.A., 1990. Migration and distribution of two populations of hippocampalgranule cell precursors during the perinatal and postnatal periods. J. Comp. Neurol.301, 365–381.

Amaral, D.G., Witter, M.P., 1995. Hippocampal formation. The Rat Nervous System, GPaxinos. Academic Press, pp. 443–492.

Amir, R.E., Van den Veyver, I.B., Wan, M., Tran, C.Q., Francke, U., Zoghbi, H.Y., 1999. Rettsyndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat. Genet. 23, 185–188.

Anthony, J.P., Sexton, T.J., Neumaier, J.F., 2000. Antidepressant-induced regulation of5-HT(1b) mRNA in rat dorsal raphe nucleus reverses rapidly after drug discontinua-tion. J. Neurosci. Res. 61, 82–87.

Babri, S., Badie, H.G., Khamenei, S., Seyedlar, M.O., 2007. Intrahippocampal insulinimproves memory in a passive-avoidance task in male Wistar rats. Brain Cogn. 64,86–91.

Banasr, M., Hery, M., Printemps, R., Daszuta, A., 2004. Serotonin-induced increases in adultcell proliferation and neurogenesis are mediated through different and common5-HT receptor subtypes in the dentate gyrus and the subventricular zone.Neuropsychopharmacology 29, 450–460.

Bar-Peled, O., Gross-Isseroff, R., Ben-Hur, H., Hoskins, I., Groner, Y., Biegon, A., 1991. Fetalhuman brain exhibits a prenatal peak in the density of serotonin 5-HT1A receptors.Neurosci. Lett. 127, 173–176.

Bartesaghi, R., Guidi, S., Ciani, E., 2011. Is it possible to improve neurodevelopmentalabnormalities in Down syndrome? Rev. Neurosci. 22, 419–455.

Bevins, R.A., Besheer, J., 2006. Object recognition in rats and mice: a one-trialnon-matching-to-sample learning task to study ‘recognition memory’. Nat. Protoc.1, 1306–1311.

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

Boullin, D.J., O'Brien, R.A., 1971. Abnormalities of 5-hydroxytryptamine uptake and bind-ing by blood platelets from children with Down's syndrome. J. Physiol. 212, 287–297.

Caccia, S., Cappi, M., Fracasso, C., Garattini, S., 1990. Influence of dose and route of admin-istration on the kinetics of fluoxetine and its metabolite norfluoxetine in the rat.Psychopharmacology (Berl) 100, 509–514.

Cai, H., Wang, Y., McCarthy, D., Wen, H., Borchelt, D.R., Price, D.L., Wong, P.C., 2001. BACE1is the major beta-secretase for generation of Abeta peptides by neurons. Nat.Neurosci. 4, 233–234.

Cassel, S., Carouge, D., Gensburger, C., Anglard, P., Burgun, C., Dietrich, J.B., Aunis, D.,Zwiller, J., 2006. Fluoxetine and cocaine induce the epigenetic factors MeCP2 andMBD1 in adult rat brain. Mol. Pharmacol. 70, 487–492.

Cataldo, A.M., Petanceska, S., Peterhoff, C.M., Terio, N.B., Epstein, C.J., Villar, A., Carlson, E.J.,Staufenbiel, M., Nixon, R.A., 2003. App gene dosage modulates endosomal abnormal-ities of Alzheimer's disease in a segmental trisomy 16 mouse model of downsyndrome. J. Neurosci. 23, 6788–6792.

Cataldo, A.M., Mathews, P.M., Boiteau, A.B., Hassinger, L.C., Peterhoff, C.M., Jiang, Y.,Mullaney, K., Neve, R.L., Gruenberg, J., Nixon, R.A., 2008. Down syndrome fibroblastmodel of Alzheimer-related endosome pathology: accelerated endocytosis promoteslate endocytic defects. Am. J. Pathol. 173, 370–384.

Chahrour, M., Jung, S.Y., Shaw, C., Zhou, X., Wong, S.T., Qin, J., Zoghbi, H.Y., 2008. MeCP2, akey contributor to neurological disease, activates and represses transcription. Science320, 1224–1229.

Choi, J.H., Berger, J.D., Mazzella, M.J., Morales-Corraliza, J., Cataldo, A.M., Nixon, R.A.,Ginsberg, S.D., Levy, E., Mathews, P.M., 2009. Age-dependent dysregulation of brainamyloid precursor protein in the Ts65Dn Down syndrome mouse model.J. Neurochem. 110, 1818–1827.

Cirrito, J.R., Disabato, B.M., Restivo, J.L., Verges, D.K., Goebel, W.D., Sathyan, A., Hayreh, D.,D'Angelo, G., Benzinger, T., Yoon, H., Kim, J., Morris, J.C., Mintun, M.A., Sheline, Y.I.,2011. Serotonin signaling is associated with lower amyloid-beta levels and plaquesin transgenic mice and humans. Proc. Natl. Acad. Sci. U. S. A. 108, 14968–14973.

Contestabile, A., Fila, T., Ceccarelli, C., Bonasoni, P., Bonapace, L., Santini, D., Bartesaghi, R.,Ciani, E., 2007. Cell cycle alteration and decreased cell proliferation in the hippocampaldentate gyrus and in the neocortical germinal matrix of fetuses with Down syndromeand in Ts65Dn mice. Hippocampus 17, 665–678.

Contestabile, A., Fila, T., Bartesaghi, R., Ciani, E., 2009. Cell cycle elongation impairs prolif-eration of cerebellar granule cell precursors in the Ts65Dn mouse, an animal modelfor Down syndrome. Brain Pathol. 19, 224–237.

Contestabile, A., Greco, B., Ghezzi, D., Tucci, V., Benfenati, F., Gasparini, L., 2013. Lithiumrescues synaptic plasticity and memory in Down syndrome mice. J. Clin. Invest.123, 348–361.

Cossec, J.C., Lavaur, J., Berman, D.E., Rivals, I., Hoischen, A., Stora, S., Ripoll, C., Mircher, C.,Grattau, Y., Olivomarin, J.C., de Chaumont, F., Lecourtois, M., Antonarakis, S.E.,Veltman, J.A., Delabar, J.M., Duyckaerts, C., Di Paolo, G., Potier, M.C., 2012. Trisomyfor synaptojanin1 in Down syndrome is functionally linked to the enlargement ofearly endosomes. Hum. Mol. Genet. 21, 3156–3172.

Costa, A.C., Scott-McKean, J.J., 2013. Prospects for improving brain function in individualswith down syndrome. CNS Drugs 27, 679–702.

Cowen, D.S., 2007. Serotonin and neuronal growth factors — a convergence of signalingpathways. J. Neurochem. 101, 1161–1171.

Crawley, J., 2007. What's Wrong With my Mouse? Wiley-Interscience - A John Wiley &Sons, Inc., Publication

DeLong, G.R., Ritch, C.R., Burch, S., 2002. Fluoxetine response in children with autisticspectrum disorders: correlation with familial major affective disorder and intellectualachievement. Dev. Med. Child Neurol. 44, 652–659.

DeVane, C.L., 1994. Pharmacokinetics of the newer antidepressants: clinical relevance.Am. J. Med. 97, 13S–23S.

218 F. Stagni et al. / Neurobiology of Disease 74 (2015) 204–218

Dierssen, M., 2012. Down syndrome: the brain in trisomic mode. Nat. Rev. Neurosci. 13,844–858.

Einarson, A., Choi, J., Einarson, T.R., Koren, G., 2009. Incidence of major malformations ininfants following antidepressant exposure in pregnancy: results of a large prospectivecohort study. Can. J. Psychiatry 54, 242–246.

Encinas, J.M., Vaahtokari, A., Enikolopov, G., 2006. Fluoxetine targets early progenitor cellsin the adult brain. Proc. Natl. Acad. Sci. U. S. A. 103, 8233–8238.

Engidawork, E., Gulesserian, T., Seidl, R., Cairns, N., Lubec, G., 2001. Expression of apoptosisrelated proteins: RAIDD, ZIP kinase, Bim/BOD, p21, Bax, Bcl-2 and NF-kappaB in brainsof patients with Down syndrome. J. Neural Transm. Suppl. 181–192.

Fernandez, F., Morishita, W., Zuniga, E., Nguyen, J., Blank, M., Malenka, R.C., Garner, C.C.,2007. Pharmacotherapy for cognitive impairment in a mouse model of Downsyndrome. Nat. Neurosci. 10, 411–413.

Gallagher, M., Burwell, R., Burchinal, M., 1993. Severity of spatial learning impairment inaging: development of a learning index for performance in the Morris water maze.Behav. Neurosci. 107, 618–626.

Ginsberg, S.D., Mufson, E.J., Alldred, M.J., Counts, S.E., Wuu, J., Nixon, R.A., Che, S., 2011.Upregulation of select rab GTPases in cholinergic basal forebrain neurons in mildcognitive impairment and Alzheimer's disease. J. Chem. Neuroanat. 42, 102–110.

Guidi, S., Bonasoni, P., Ceccarelli, C., Santini, D., Gualtieri, F., Ciani, E., Bartesaghi, R.,2008. Neurogenesis impairment and increased cell death reduce total neuronnumber in the hippocampal region of fetuses with Down syndrome. BrainPathol. 18, 180–197.

Guidi, S., Ciani, E., Bonasoni, P., Santini, D., Bartesaghi, R., 2011. Widespread proliferationimpairment and hypocellularity in the cerebellum of fetuses with Down syndrome.Brain Pathol. 21, 361–373.

Guidi, S., Stagni, F., Bianchi, P., Ciani, E., Ragazzi, E., Trazzi, S., Grossi, G., Mangano, C., Calza, L.,Bartesaghi, R., 2013. Early pharmacotherapy with fluoxetine rescues dendriticpathology in the Ts65Dnmousemodel of Down syndrome. Brain Pathol. 23, 129–143.

Guidi, S., Stagni, F., Bianchi, P., Ciani, E., Giacomini, A., De Franceschi, M., Moldrich, R.,Kurniawan, N., Mardon, K., Giuliani, A., Calza, L., Bartesaghi, R., 2014. Prenatalpharmacotherapy rescues brain development in a Down's syndrome mouse model.Brain 137, 380–401.

Hayes, R.M., Wu, P., Shelton, R.C., Cooper, W.O., Dupont, W.D., Mitchel, E., Hartert, T.V.,2012. Maternal antidepressant use and adverse outcomes: a cohort study of228,876 pregnancies. Am. J. Obstet. Gynecol. 207 (49), e41–e49.

Hollander, E., Phillips, A., Chaplin,W., Zagursky, K., Novotny, S., Wasserman, S., Iyengar, R.,2005. A placebo controlled crossover trial of liquid fluoxetine on repetitive behaviorsin childhood and adolescent autism. Neuropsychopharmacology 30, 582–589.

Huang, G.J., Ben-David, E., Tort Piella, A., Edwards, A., Flint, J., Shifman, S., 2012.Neurogenomic evidence for a shared mechanism of the antidepressant effects ofexercise and chronic fluoxetine in mice. PLoS ONE 7, e35901.

Hunter, C.L., Bimonte, H.A., Granholm, A.C., 2003a. Behavioral comparison of 4 and6 month-old Ts65Dn mice: age-related impairments in working and referencememory. Behav. Brain Res. 138, 121–131.

Hunter, C.L., Isacson, O., Nelson,M., Bimonte-Nelson, H., Seo, H., Lin, L., Ford, K., Kindy, M.S.,Granholm, A.C., 2003b. Regional alterations in amyloid precursor protein and nervegrowth factor across age in a mouse model of Down's syndrome. Neurosci. Res. 45,437–445.

Jiang, Y., Mullaney, K.A., Peterhoff, C.M., Che, S., Schmidt, S.D., Boyer-Boiteau, A., Ginsberg,S.D., Cataldo, A.M., Mathews, P.M., Nixon, R.A., 2009. Alzheimer's-related endosomedysfunction in Down syndrome is Abeta-independent but requires APP and isreversed by BACE-1 inhibition. Proc. Natl. Acad. Sci. U. S. A. 107, 1630–1635.

Kuhn, D.E., Nuovo, G.J., Terry Jr., A.V., Martin, M.M., Malana, G.E., Sansom, S.E., Pleister,A.P., Beck, W.D., Head, E., Feldman, D.S., Elton, T.S., 2010. Chromosome 21-derivedmicroRNAs provide an etiological basis for aberrant protein expression in humanDown syndrome brains. J. Biol. Chem. 285, 1529–1543.

Le Poul, E., Boni, C., Hanoun, N., Laporte, A.M., Laaris, N., Chauveau, J., Hamon, M.,Lanfumey, L., 2000. Differential adaptation of brain 5-HT1A and 5-HT1B receptorsand 5-HT transporter in rats treated chronically with fluoxetine. Neuropharmacology39, 110–122.

Liu, C., Belichenko, P.V., Zhang, L., Fu, D., Kleschevnikov, A.M., Baldini, A., Antonarakis, S.E.,Mobley, W.C., Yu, Y.E., 2011. Mouse models for Down syndrome-associated develop-mental cognitive disabilities. Dev. Neurosci. 33, 404–413.

Lockrow, J., Boger, H., Bimonte-Nelson, H., Granholm, A.C., 2010. Effects of long-termmemantine on memory and neuropathology in Ts65Dn mice, a model for Downsyndrome. Behav. Brain Res. 221, 610–622.

Loizzo, S., Rimondini, R., Travaglione, S., Fabbri, A., Guidotti, M., Ferri, A., Campana, G.,Fiorentini, C., 2013. CNF1 increases brain energy level, counteracts neuroinflammatorymarkers and rescues cognitive deficits in a murine model of Alzheimer's disease.PLoS ONE 8, e65898.

Lott, I.T., Chase, T.N., Murphy, D.L., 1972a. Down's syndrome: transport, storage, andmetabolism of serotonin in blood platelets. Pediatr. Res. 6, 730–735.

Lott, I.T., Murphy, D.L., Chase, T.N., 1972b. Down's syndrome. Central monoamineturnover in patients with diminished platelet serotonin. Neurology 22, 967–972.

Ly, P.T., Wu, Y., Zou, H., Wang, R., Zhou, W., Kinoshita, A., Zhang, M., Yang, Y., Cai, F.,Woodgett, J., Song, W., 2013. Inhibition of GSK3beta-mediated BACE1 expressionreduces Alzheimer-associated phenotypes. J. Clin. Invest. 123, 224–235.

Lyle, R., Gehrig, C., Neergaard-Henrichsen, C., Deutsch, S., Antonarakis, S.E., 2004. Geneexpression from the aneuploid chromosome in a trisomy mouse model of downsyndrome. Genome Res. 14, 1268–1274.

Mogha, A., Guariglia, S.R., Debata, P.R., Wen, G.Y., Banerjee, P., 2012. Serotonin 1Areceptor-mediated signaling through ERK and PKCalpha is essential for normalsynaptogenesis in neonatal mouse hippocampus. Transl. Psychiatry 2, e66.

Nelson, R.L., Guo, Z., Halagappa, V.M., Pearson, M., Gray, A.J., Matsuoka, Y., Brown, M.,Martin, B., Iyun, T., Maudsley, S., Clark, R.F., Mattson, M.P., 2007. Prophylactic treat-ment with paroxetine ameliorates behavioral deficits and retards the developmentof amyloid and tau pathologies in 3xTgAD mice. Exp. Neurol. 205, 166–176.

Netzer, W.J., Powell, C., Nong, Y., Blundell, J., Wong, L., Duff, K., Flajolet, M., Greengard, P.,2010. Lowering beta-amyloid levels rescues learning and memory in a Downsyndrome mouse model. PLoS ONE 5, e10943.

Nowakowski, R.S., Lewin, S.B., Miller, M.W., 1989. Bromodeoxyuridine immunohisto-chemical determination of the lengths of the cell cycle and the DNA-syntheticphase for an anatomically defined population. J. Neurocytol. 18, 311–318.

Olivier, J., Akerud, H., Kaihola, L., Pawluski, J., Skalkidou, S., Hogberg, U., Sundstrom-Poromaa, I., 2013. The Effects ofMaternal Depression andMaternal Selective SerotoninReuptake Inhibitor Exposure on Offspring. 7. http://dx.doi.org/10.3389/fncel.2013.00073.

Park, J., Oh, Y., Yoo, L., Jung, M.S., Song, W.J., Lee, S.H., Seo, H., Chung, K.C., 2010. Dyrk1Aphosphorylates p53 and inhibits proliferation of embryonic neuronal cells. J. Biol.Chem. 285, 31895–31906.

Pechnick, R.N., Zonis, S.,Wawrowsky, K., Cosgayon, R., Farrokhi, C., Lacayo, L., Chesnokova, V.,2011. Antidepressants stimulate hippocampal neurogenesis by inhibiting p21expression in the subgranular zone of the hipppocampus. PLoS ONE 6, e27290.

Pedersen, L.H., Henriksen, T.B., Olsen, J., 2010. Fetal exposure to antidepressants andnormal milestone development at 6 and 19 months of age. Pediatrics 125,e600–e608.

Reeves, R.H., Irving, N.G., Moran, T.H., Wohn, A., Kitt, C., Sisodia, S.S., Schmidt, C., Bronson,R.T., Davisson, M.T., 1995. A mouse model for Down syndrome exhibits learning andbehaviour deficits. Nat. Genet. 11, 177–184.

Reinholdt, L.G., Ding, Y., Gilbert, G.J., Czechanski, A., Solzak, J.P., Roper, R.J., Johnson, M.T.,Donahue, L.R., Lutz, C., Davisson, M.T., 2011. Molecular characterization of thetranslocation breakpoints in the Down syndrome mouse model Ts65Dn. Mamm.Genome 22, 685–691.

Rhin, L.L., Claiborne, B.J., 1990. Dendritic growth and regression in rat dentate granulecells during late postnatal development. Dev. Brain Res. 54, 115–124.

Riekkinen Jr., P., Riekkinen,M., Sirvio, J., 1993. Cholinergic drugs regulate passive avoidanceperformance via the amygdala. J. Pharmacol. Exp. Ther. 267, 1484–1492.

Roper, R.J., St John, H.K., Philip, J., Lawler, A., Reeves, R.H., 2006. Perinatal loss of TS65DNDown syndrome mice. Genetics 172, 437–443.

Sairanen, M., Lucas, G., Ernfors, P., Castren, M., Castren, E., 2005. Brain-derived neuro-trophic factor and antidepressant drugs have different but coordinated effects onneuronal turnover, proliferation, and survival in the adult dentate gyrus. J. Neurosci.25, 1089–1094.

Salehi, A., Faizi, M., Colas, D., Valletta, J., Laguna, J., Takimoto-Kimura, R., Kleschevnikov, A.,Wagner, S.L., Aisen, P., Shamloo, M., Mobley, W.C., 2009. Restoration ofnorepinephrine-modulated contextual memory in a mouse model of Downsyndrome. Sci. Transl. Med. 1, 7ra17.

Santarelli, L., Saxe, M., Gross, C., Surget, A., Battaglia, F., Dulawa, S., Weisstaub, N., Lee, J.,Duman, R., Arancio, O., Belzung, C., Hen, R., 2003. Requirement of hippocampalneurogenesis for the behavioral effects of antidepressants. Science 301, 805–809.

Schlessinger, A.R., Cowan, W.M., Gottlieb, D.I., 1975. An autoradiographic study of thetime of origin and the pattern of granule cell migration in the dentate gyrus of therat. J. Comp. Neurol. 159, 149–175.

Seo, H., Isacson, O., 2005. Abnormal APP, cholinergic and cognitive function in Ts65DnDown's model mice. Exp. Neurol. 193, 469–480.

Stagni, F., Magistretti, J., Guidi, S., Ciani, E., Mangano, C., Calza, L., Bartesaghi, R., 2013.Pharmacotherapy with fluoxetine restores functional connectivity from the dentategyrus to Field CA3 in the Ts65Dn mouse model of Down syndrome. PLoS ONE 8,e61689.

Sultan, M., Piccini, I., Balzereit, D., Herwig, R., Saran, N.G., Lehrach, H., Reeves, R.H., Yaspo,M.L., 2007. Gene expression variation in Down's syndrome mice allows prioritizationof candidate genes. Genome Biol. 8, R91.

Sun, X., Tong, Y., Qing, H., Chen, C.H., Song,W., 2006. Increased BACE1maturation contrib-utes to the pathogenesis of Alzheimer's disease in Down syndrome. FASEB J. 20,1361–1368.

Thomas, G.M., Huganir, R.L., 2004. MAPK cascade signalling and synaptic plasticity. Nat.Rev. Neurosci. 5, 173–183.

Trazzi, S., Mitrugno, V.M., Valli, E., Fuchs, C., Rizzi, S., Guidi, S., Perini, G., Bartesaghi, R.,Ciani, E., 2011. APP-dependent up-regulation of Ptch1 underlies proliferation impair-ment of neural precursors in Down syndrome. Hum. Mol. Genet. 20, 1560–1573.

Trazzi, S., Fuchs, C., De Franceschi, M., Mitrugno, V., Bartesaghi, R., Ciani, E., 2014. APP-dependent alteration of GSK3ß activity impairs neurogenesis in the Ts65Dn mousemodel of Down syndrome. Neurobiol. Dis. http://dx.doi.org/10.1016/j.nbd.2014.03.003 (Epub ahead of print).

Whitaker-Azmitia, P.M., 2001. Serotonin and brain development: role in human develop-mental diseases. Brain Res. Bull. 56, 479–485.

Whittle, N., Sartori, S.B., Dierssen, M., Lubec, G., Singewald, N., 2007. Fetal Down syndromebrains exhibit aberrant levels of neurotransmitters critical for normal brain develop-ment. Pediatrics 120, e1465–e1471.

Yan, W., Wilson, C.C., Haring, J.H., 1997a. 5-HT1a receptors mediate the neurotrophiceffect of serotonin on developing dentate granule cells. Brain Res. Dev. Brain Res.98, 185–190.

Yan, W., Wilson, C.C., Haring, J.H., 1997b. Effects of neonatal serotonin depletion on thedevelopment of rat dentate granule cells. Brain Res. Dev. Brain Res. 98, 177–184.

Zhao, C., Teng, E.M., Summers Jr., R.G., Ming, G.L., Gage, F.H., 2006. Distinct morphologicalstages of dentate granule neuron maturation in the adult mouse hippocampus.J. Neurosci. 26, 3–11.