Prenatal treatment with the neuroprotective peptides NAP and SAL prevented the learning deficits in adult mice, with learning similar to control animals. Our findings are novel in that we propose the possibility of an intervention during pregnancy that may prevent or reverse the learning deficits of DS. These results extend the benefit shown with prenatal treatment in the Ts65Dn model where NAP and SAL prevented developmental delay in the DS offspring and with postnatal treatment where NAP and SAL prevented learning deficit in Ts65Dn animals treated during adulthood 
. The results are consistent with studies of learning enhancement with NAP and SAL from our group in a model of fetal alcohol syndrome 
and in normal animals 
. Others have also shown that neonatal mice treated with intraperitoneal NAP had increased performance in the Morris watermaze and that VIP is dysregulated in the Ts65Dn partial trisomy model for DS 
These results may, at least in part, be explained by our findings of alterations in the N-methyl-D-aspartate (NMDA) receptors. Using this DS mouse model we have shown that prenatal administration of NAP and SAL during pregnancy has long-lasting effects, specifically increasing NR2B and GABAAα3 expression in adult Ts65Dn mice to levels similar to wild-type controls. In the NMDA receptors, the NR2B receptor is the more plastic subunit, thus a decrease in NR2B would make the synapse less plastic. Given the interaction between the GABA and NMDA receptors, a decrease in GABAAα3 would provide less inhibitory tone on the synapse causing overexcitation and ultimately termination of this neural synapse. Restoring the levels of these receptors to the level in controls may in part explain how the peptides NAP and SAL prevent adult learning impairment in the mouse model 
. Fernandez et al. 
demonstrated that chronic systemic treatment of Ts65Dn mice with GABAA antagonists causes a persistent post-drug recovery of cognition and LTP. The results support the hypothesis that excessive GABA-mediated inhibition in the Ts65Dn brain actively interferes with declarative memory in Ts65Dn mice 
Regarding the protective effects of the peptides on learning and memory, previous studies of hippocampal cultures demonstrated that the peptides alter glutamate release and NMDA receptors 
, both of which are important in learning and memory. Particularly, treatment of hippocampal neurons with SAL controls NR2A and NR2B subunit stability of the NMDA receptor in neurons that have yet to establish efficient synaptic connections 
. Although the exact mechanism by which SAL influences the synaptogenesis and neurotransmission used by glutamate remains unknown, in vitro studies strongly suggest that the peptide interacts with and regulates the glutaminergic synapses in developing neural systems. Kleschevnikov et al. found that LTP could not be elicited in the dentate gyrus of Ts65Dn mice. They suggested that excessive inhibition of dentate granule cells was shown to restrict synaptic activation of NMDA receptors and to inhibit LTP 
One of the neuropathologic characteristics of DS is a glial deficit that likely induces alterations in VIP and its related neuropeptides. In the Ts65Dn mouse, we and others have showen a decrease in ADNP and an upregulation of VIP 
. Prenatal treatment with NAL and SAL resulted in a normalization of ADNP and of the glial marker GFAP in Ts65Dn adult brains 
. Treatment with the peptides may have overcome the glial deficit by restoring the appropriate neuropeptides. Nonetheless, treatment did not prevent VIP upregulation; it is possible that other mechanisms regulate VIP release 
Previous studies have shown that ADNF and ADNP are neuroprotective in the presence of toxins associated with neurodegenerative disorders, and that neonatal treatment with ADNP attenuated head injury–related dysfunctions in adulthood 
. Numerous studies have shown the protective effects of VIP-related peptides against alcohol developmental effects 
. Here we confirm the role of NAP and SAL in the prevention of learning impairment in a model of DS.
The strengths of this work include the study design and the validation of the results in three different sets of animals. Moreover, this work confirms that the Ts65Dn is a model that is particularly useful in understanding the biological basis for some of the developmental abnormalities and learning deficits of DS subjects.
The high mortality of the DS animals reduced the number of animals available, and consequently the pathway to test to understand the mechanism of action of the VIP related peptides. We encourage further studies to delineate pathways underlying the mechanism behind learning enhancement in the Ts65Dn mouse model of Down syndrome.
In conclusion, these findings highlight a possibility for the prevention of sequelae in DS and other conditions with learning deficit. Because DS can be diagnosed prenatally, an intervention during pregnancy that may improve cognitive function is an attractive option.