Physiological studies indicate that the piriform or primary olfactory cortex of adult mammals exhibits a high degree of synaptic plasticity. Interestingly, a subpopulation of cells in the layer II of the adult piriform cortex expresses neurodevelopmental markers, such as the polysialylated form of neural cell adhesion molecule (PSA-NCAM) or doublecortin (DCX). This study analyzes the nature, origin, and potential function of these poorly understood cells in mice. As previously described in rats, most of the PSANCAM expressing cells in layer II could be morphologically classified as tangled cells and only a small proportion of larger cells could be considered semilunar-pyramidal transitional neurons. Most were also immunoreactive for DCX, confirming their immature nature. In agreement with this, detection of PSA-NCAM combined with that of different cell lineage-specific antigens revealed that most PSA-NCAM positive cells did not co-express markers of glial cells or mature neurons. Their time of origin was evaluated by birthdating experiments with halogenated nucleosides performed at different developmental stages and in adulthood. We found that virtually all cells in this paleocortical region, including PSA-NCAM-positive cells, are born during fetal development. In addition, proliferation analyses in adult mice revealed that very few cells were cycling in layer II of the piriform cortex and that none of them was PSA-NCAMpositive. Moreover, we have established conditions to isolate and culture these immature neurons in the adult piriform cortex layer II. We find that although they can survive under certain conditions, they do not proliferate in vitro either. On the other hand, these cells progressively disappear along the course of aging, while their fate and function remain unclear. Thus, using DCX-CreERT2/Flox-EGFP transgenic mice, we demonstrate that these immature neurons located in the murine piriform cortex do not vanish in the course of aging, but progressively resume their maturation into glutamatergic (TBR1+, CaMKII+) neurons. We provide evidence for a putative functional integration of these newly differentiated neurons as indicated by the increase in perisomatic puncta expressing synaptic markers, the development of complex apical dendrites decorated with numerous spines and the appearance of an axonal initial segment. Since immature neurons found in layer II of the piriform cortex are generated prenatally and devoid of proliferative capacity in the postnatal cortex, the gradual maturation and integration of these cells outside of the canonical neurogenic niches implies that they represent a valuable, but nonrenewable reservoir for cortical plasticity. Finally, as PSA-NCAM confers to the cells anti-adhesive properties and limites the membrane space available for the establishment of synaptic contacts, in order to understand the role that PSA-NCAM may play in the development of the immature neurons in the adult piriform cortex layer II, we have depleted PSA from NCAM in this region using the enzyme EndoN. We found that after EndoN injection the number of immature neurons expressing doublecortin decreased in layer II. Moreover, the percentage of tangled cells diminished, while that of complex cells was increased. The depletion of PSA-NCAM from the piriform cortex also increased the density of nuclei expressing NeuN, a mature neuronal marker, in layer II. This increase was paralleled by increases in the expression of the synaptic marker synaptophysin and MAP2, a marker of mature dendrites, in layer I. Together, these results indicate a pro-maturation effect of PSA depletion. We have also tested the effect of the absence of PSA through genetic deletions of the both polysialyltransferases that can add PSA to the NCAM (STX and PST) or NCAM. The deletion of both polysialyltransferases decreases dramatically the number of immature doublecortin expressing neurons in the adult piriform cortex. By contrast, the deletion of NCAM, increases this number, suggesting the presence of compensatory effects.

Physiological studies indicate that the piriform or primary olfactory cortex of adult mammals exhibits a high degree of synaptic plasticity. Interestingly, a subpopulation of cells in the layer II of the adult piriform cortex expresses neurodevelopmental markers, such as the polysialylated form of neural cell adhesion molecule (PSA-NCAM) or doublecortin (DCX). This study analyzes the nature, origin, and potential function of these poorly understood cells in mice. As previously described in rats, most of the PSANCAM expressing cells in layer II could be morphologically classified as tangled cells and only a small proportion of larger cells could be considered semilunar-pyramidal transitional neurons. Most were also immunoreactive for DCX, confirming their immature nature. In agreement with this, detection of PSA-NCAM combined with that of different cell lineage-specific antigens revealed that most PSA-NCAM positive cells did not co-express markers of glial cells or mature neurons. Their time of origin was evaluated by birthdating experiments with halogenated nucleosides performed at different developmental stages and in adulthood. We found that virtually all cells in this paleocortical region, including PSA-NCAM-positive cells, are born during fetal development. In addition, proliferation analyses in adult mice revealed that very few cells were cycling in layer II of the piriform cortex and that none of them was PSA-NCAMpositive. Moreover, we have established conditions to isolate and culture these immature neurons in the adult piriform cortex layer II. We find that although they can survive under certain conditions, they do not proliferate in vitro either. On the other hand, these cells progressively disappear along the course of aging, while their fate and function remain unclear. Thus, using DCX-CreERT2/Flox-EGFP transgenic mice, we demonstrate that these immature neurons located in the murine piriform cortex do not vanish in the course of aging, but progressively resume their maturation into glutamatergic (TBR1+, CaMKII+) neurons. We provide evidence for a putative functional integration of these newly differentiated neurons as indicated by the increase in perisomatic puncta expressing synaptic markers, the development of complex apical dendrites decorated with numerous spines and the appearance of an axonal initial segment. Since immature neurons found in layer II of the piriform cortex are generated prenatally and devoid of proliferative capacity in the postnatal cortex, the gradual maturation and integration of these cells outside of the canonical neurogenic niches implies that they represent a valuable, but nonrenewable reservoir for cortical plasticity. Finally, as PSA-NCAM confers to the cells anti-adhesive properties and limites the membrane space available for the establishment of synaptic contacts, in order to understand the role that PSA-NCAM may play in the development of the immature neurons in the adult piriform cortex layer II, we have depleted PSA from NCAM in this region using the enzyme EndoN. We found that after EndoN injection the number of immature neurons expressing doublecortin decreased in layer II. Moreover, the percentage of tangled cells diminished, while that of complex cells was increased. The depletion of PSA-NCAM from the piriform cortex also increased the density of nuclei expressing NeuN, a mature neuronal marker, in layer II. This increase was paralleled by increases in the expression of the synaptic marker synaptophysin and MAP2, a marker of mature dendrites, in layer I. Together, these results indicate a pro-maturation effect of PSA depletion. We have also tested the effect of the absence of PSA through genetic deletions of the both polysialyltransferases that can add PSA to the NCAM (STX and PST) or NCAM. The deletion of both polysialyltransferases decreases dramatically the number of immature doublecortin expressing neurons in the adult piriform cortex. By contrast, the deletion of NCAM, increases this number, suggesting the presence of compensatory effects.