Down syndrome (DS) is caused by the triplication of human chromosome 21, with a frequency of 1/700 live births. Cognitive impairment is the most striking clinical DS feature, further worsened by several neurological co-morbidities. One of the most common co-morbidities is epilepsy, with a prevalence ranging from 1 to 13%, which is higher than in the general population. Hyperactivity can also be present with a prevalence reaching 43%. The onset of the first seizure in DS people show a bimodal distribution: in 40% of subjects it is before the first year of age and in another 40% is over 30 years of age. In DS infants, the most common type of seizures is represented by infantile spasms (IS) leading to several long-term complications (difficulties with ambulation, autistic traits, cognitive impairment). In adult DS subjects, the most common types of seizures are represented by partial or tonic-clonic seizures, with a high incidence of triggered tonic-clonic seizures (in particular audiogenic seizures, AGS). Diverse studies successfully induced seizures in DS mouse models (mostly the widely used Ts65Dn mice), recapitulating the phenotypes observed in humans. Nevertheless, the mechanisms underlying the increased susceptibility to both early and late onset seizure and to hyperactivity in DS have not yet been thoroughly studied, thus leading to poor therapeutic strategy. Interestingly, several studies indicate that a depolarizing shift of the GABAergic transmission (through Cl-permeable GABAA receptors) due to increased Cl concentration inside neurons, is a common mechanism underlying several brain disorders including DS and epilepsy. Cl is mainly regulated by the action of two Cl transporters, namely NKCC1 and KCC2. In line with the dysregulated Cl concentration, NKCC1/KCC2 expression ratio is impaired in Ts65Dn mice and rodent models of epilepsy. In particular, in our lab we found that NKCC1 is upregulated in the hippocampus and cortex of adult Ts65Dn mice and DS people. Interestingly NKCC1 inhibition by bumetanide, rescues memory deficits in adult animals. However, bumetanide treatment in adult Ts65Dn mice failed to rescue their higher susceptibility to AGS and the hyperactivity, although bumetanide showed to be beneficial in rescuing seizures in adult rodent models of other types of epilepsy (e.g. temporal lobe epilepsy) and in clinical trials. Moreover, the positive effects of bumetanide in memory tasks were lost after one week of drug withdrawal, indicating that bumetanide treatment in adulthood did not induce neuronal rewiring. Altogether, these results suggest that the increased susceptibility to seizures and hyperactivity in Ts65Dn mice might depend on a miswiring of the brain circuits and not on the solely alteration of Cl- homeostasis per se in adulthood. Thus, in this project we will investigate whether an alteration of intracellular Cl concentration early in development can contribute to inducing a miswiring of the brain circuits, which consequently lead to neurological impairment (i.e., increased seizure susceptibility, hyperactivity) lasting lifelong. In this scenario, we will assess if a timely, and early pharmacological NKCC1 inhibition acting when neuronal networks are still plastic could ameliorate symptoms that treatments in adult ages were not able to reverse.