Adult neurogenesis occurs in the hippocampus and the olfactory bulb of the mammalian CNS. Recent studies have demonstrated that newborn granule cells of the adult hippocampus are postsynaptic targets of excitatory and inhibitory neurons, but evidence of synapse formation by the axons of these cells is still lacking. By combining retroviral expression of green fluorescent protein in adult-born neurons of the mouse dentate gyrus with immuno-electron microscopy, we found output synapses that were formed by labeled terminals on appropriate target cells in the CA3 area and the hilus. Furthermore, retroviral expression of channelrhodopsin-2 allowed us to light-stimulate newborn granule cells and identify postsynaptic target neurons by whole-cell recordings in acute slices. Our structural and functional evidence indicates that axons of adult-born granule cells establish synapses with hilar interneurons, mossy cells and CA3 pyramidal cells and release glutamate as their main neurotransmitter.Increasing evidence supports the hypothesis that neurogenesis is a physiologically important event in the adult hippocampus, but the precise role of newly generated cells in the hippocampal network remains unknown 1-5 . Understanding how newborn granule cells may contribute to information processing in the adult hippocampus requires a detailed analysis of their connectivity and function. Previous studies have clearly demonstrated that newborn neurons in the adult hippocampus receive morphologically mature axo-somatic, axo-dendritic and axospinous synapses and that those inputs arising from the entorhinal cortex and local inhibitory interneurons are fully functional 6-13 . To influence information processing in the hippocampal network, new granule cells must also contact the appropriate neuronal targets, but this capability has not yet been demonstrated because of technical challenges.Correspondence should be addressed to F.H.G. (gage@salk.edu) or A.F.S. (aschinder@leloir.org.ar). 4 These authors contributed equally to this work. AUTHOR CONTRIBUTIONS N.T. contributed to the concept, designed and carried out the structural experiments, analyzed the data, and wrote the manuscript. D.A.L. contributed to the concept, designed and performed the functional experiments, analyzed the data, and wrote the manuscript. C.Z. contributed to the experimental design, provided samples for the structural experiments, carried out and analyzed confocal images of presynaptic terminals, and revised the manuscript. G.L. prepared retroviral stocks, performed immunofluorescence and obtained images of ChR2-positive neurons. C.E.R. contributed to setting up the technique for immuno-electron microscopy of GFP, the analysis of electron micrographs, and the writing and revision of the manuscript. F.H.G. and A.F.S. contributed to the concept, designed the experiments, analyzed the data, wrote the manuscript and provided financial support. In this study, we searched for structural and functional evidence for output synapses of adultborn granule cells. Mossy...

Neurons born in the adult dentate gyrus develop, mature, and connect over a long interval that can last from six to eight weeks. It has been proposed that, during this period, developing neurons play a relevant role in hippocampal signal processing owing to their distinctive electrical properties. However, it has remained unknown whether immature neurons can be recruited into a network before synaptic and functional maturity have been achieved. To address this question, we used retroviral expression of green fluorescent protein to identify developing granule cells of the adult mouse hippocampus and investigate the balance of afferent excitation, intrinsic excitability, and firing behavior by patch clamp recordings in acute slices. We found that glutamatergic inputs onto young neurons are significantly weaker than those of mature cells, yet stimulation of cortical excitatory axons elicits a similar spiking probability in neurons at either developmental stage. Young neurons are highly efficient in transducing ionic currents into membrane depolarization due to their high input resistance, which decreases substantially in mature neurons as the inward rectifier potassium (Kir) conductance increases. Pharmacological blockade of Kir channels in mature neurons mimics the high excitability characteristic of young neurons. Conversely, Kir overexpression induces mature-like firing properties in young neurons. Therefore, the differences in excitatory drive of young and mature neurons are compensated by changes in membrane excitability that render an equalized firing activity. These observations demonstrate that the adult hippocampus continuously generates a population of highly excitable young neurons capable of information processing.

The dentate gyrus of the hippocampus generates neurons throughout life, but adult neurogenesis exhibits a marked age-dependent decline. Although the decrease in the rate of neurogenesis has been extensively documented in the ageing hippocampus, the specific characteristics of dentate granule cells born in such a continuously changing environment have received little attention. We have used retroviral labelling of neural progenitor cells of the adult mouse dentate gyrus to study morphological properties of neurons born at different ages. Dendritic spine density was measured to estimate glutamatergic afferent connectivity. Fully mature neurons born at the age of 2 months display ∼2.3 spines μm −1 and maintain their overall morphology and spine density in 1-year-old mice. Surprisingly, granule cells born in 10-month-old mice, at which time the rate of neurogenesis has decreased by ∼40-fold, reach a density of dendritic spines similar to that of neurons born in young adulthood. Therefore, in spite of the sharp decline in cell proliferation, differentiation and overall neuronal number, the ageing hippocampus presents a suitable environment for new surviving neurons to reach a high level of complexity, comparable to that of all other dentate granule cells.