Down syndrome (DS), the leading genetic cause of mental retardation, is characterized by reduced number of cortical neurons and brain size. The occurrence of these defects starting from early life stages points at altered developmental neurogenesis as their major determinant. The goal of our study was to obtain comparative evidence for impaired neurogenesis in the hippocampal dentate gyrus (DG) of DS fetuses and Ts65Dn mice, an animal model for DS. Cell proliferation in human fetuses was evaluated with Ki-67 (a marker of cells in S + G(2) + M phases of cell cycle) and cyclin A (a marker of cells in S phase) immunohistochemistry. We found that in the DG of DS fetuses the number of proliferating cells was notably reduced when compared with controls. A similar reduction was observed in the germinal matrix of the lateral ventricle. In both structures, DS fetuses showed a reduced ratio between cyclin A- and Ki-67-positive cells when compared with controls, indicating that they had a reduced number of cycling cells in S phase. In the DG of P2 Ts65Dn mice cell proliferation, assessed 2 h after an injection of bromodeoxyuridine (BrdU), was notably reduced, similarly to DS fetuses. After 28 days, Ts65Dn mice had still less BrdU-positive cells than controls. Phenotypic analysis of the surviving cells showed that Ts65Dn mice had a percent number of cells with astrocytic phenotype larger than controls. Using phospho-histone H3 immunohistochemistry we found that both DS fetuses and P2 Ts65Dn mice had a higher number of proliferating cells in G(2) and a smaller number of cells in M phase of cell cycle. Results provide novel evidence for proliferation impairment in the hippocampal DG of the DS fetal brain, comparable to that of the P2 mouse model, and suggest that cell cycle alterations may be critical determinants of the reduced proliferation potency.

We previously obtained evidence for reduced cell proliferation in the dentate gyrus (DG) of fetuses with Down syndrome (DS), suggesting that the hippocampal hypoplasia seen in adulthood may be caused by defective early neuron production. The goal of this study was to establish whether DS fetuses (17-21 weeks of gestation) exhibit reduction in total cell number in the DG, hippocampus and parahippocampal gyrus (PHG). Volumes of the cellular layers and cell number were estimated with Cavalieri's principle and the optical fractionator method, respectively. We found that in DS fetuses all investigated structures had a reduced volume and cell number. Analysis of cell phenotype showed that DS fetuses had a higher percentage of cells with astrocytic phenotype but a smaller percentage of cells with neuronal phenotype. Immunohistochemistry for Ki-67, a marker of cycling cells, showed that DS fetuses had less proliferating cells in the germinal zones of the hippocampus and PHG. We additionally found that in the hippocampal region of DS fetuses there was a higher incidence of apoptotic cell death. Results show reduced neuron number in the DS hippocampal region and suggest that this defect is caused by disruption of neurogenesis and apoptosis, two fundamental processes underlying brain building.

Marijuana and hashish are the illicit drugs most frequently used by human adolescents. Given the continued neurodevelopment throughout adolescence, adolescents may be more vulnerable than adults to certain neural consequences of heavy marijuana use. This study aimed to assess whether an experimental model of adolescent chronic exposure to Delta9-tetrahydrocannabinol (THC), may induce lasting effects on learning and memory. Adolescent rats have been treated with THC or its vehicle from 35 to 45 postnatal days (PND) and left undisturbed until their adulthood (75 PND) when aversive and spatial memory was assessed using the passive avoidance and radial maze tasks. No alteration was found in aversive memory, but in the radial maze THC pretreated animals exhibited a worse performance than vehicles, suggesting a deficit in spatial working memory. To correlate memory impairment to altered neuroplasticity, level of marker proteins was investigated in the hippocampus, the most relevant area mediating spatial memory. A significant decrease in the astroglial marker glial fibrillar acid protein was found as well as in pre- and postsynaptic protein expression (VAMP2, PSD95) and NMDA receptor levels in pretreated rats. To parallel these changes to alteration in dendritic morphology, Golgi-Cox staining was performed in the hippocampal dentate gyrus. Pretreated rats had a significantly lower total dendritic length and number than vehicles, as well as reduced spine density. Our data suggest that THC pretreated rats may establish less synaptic contacts and/or less efficient synaptic connections throughout the hippocampus and this could represent the molecular underpinning of the cognitive deficit induced by adolescent THC treatment.

Down syndrome (DS) is a genetic pathology characterized by intellectual disability and brain hypotrophy. Widespread neurogenesis impairment characterizes the fetal and neonatal DS brain, strongly suggesting that this defect may be a major determinant of mental retardation. Our goal was to establish, in a mouse model for DS, whether early pharmacotherapy improves neurogenesis and cognitive behavior. Neonate Ts65Dn mice were treated from postnatal day (P) 3 to P15 with fluoxetine, an antidepressant that inhibits serotonin (5-HT) reuptake and increases proliferation in the adult Ts65Dn mouse (Clark et al., 2006). On P15, they received a BrdU injection and were killed after either 2 h or 1 month. Results showed that P15 Ts65Dn mice had notably defective proliferation in the hippocampal dentate gyrus, subventricular zone, striatum, and neocortex and that proliferation was completely rescued by fluoxetine. In the hippocampus of untreated P15 Ts65Dn mice, we found normal 5-HT levels but a lower expression of 5-HT1A receptors and brain-derived neurotrophic factor (BDNF). In Ts65Dn mice, fluoxetine treatment restored the expression of 5-HT1A receptors and BDNF. One month after cessation of treatment, there were more surviving cells in the dentate gyrus of Ts65Dn mice, more cells with a neuronal phenotype, more proliferating precursors, and more granule cells. These animals were tested for contextual fear conditioning, a hippocampusdependent memory task, and exhibited a complete recovery of memory performance. Results show that early pharmacotherapy with a drug usable by humans can correct neurogenesis and behavioral impairment in a model for DS.

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in a neurodevelopmental disorder characterized by early-onset intractable seizures, severe developmental delay, intellectual disability, and Rett's syndrome-like features. Since the physiological functions of CDKL5 still need to be elucidated, in the current study we took advantage of a new Cdkl5 knockout (KO) mouse model in order to shed light on the role of this gene in brain development. We mainly focused on the hippocampal dentate gyrus, a region that largely develops postnatally and plays a key role in learning and memory. Looking at the process of neurogenesis, we found a higher proliferation rate of neural precursors in Cdkl5 KO mice in comparison with wild type mice. However, there was an increase in apoptotic cell death of postmitotic granule neuron precursors, with a reduction in total number of granule cells. Looking at dendritic development, we found that in Cdkl5 KO mice the newly-generated granule cells exhibited a severe dendritic hypotrophy. In parallel, these neurodevelopmental defects were associated with impairment of hippocampus-dependent memory. Looking at the mechanisms whereby CDKL5 exerts its functions, we identified a central role of the AKT/GSK-3β signaling pathway. Overall our findings highlight a critical role of CDKL5 in the fundamental processes of brain development, namely neuronal precursor proliferation, survival and maturation. This evidence lays the basis for a better understanding of the neurological phenotype in patients carrying mutations in the CDKL5 gene.