The present study demonstrates that, in young adult mice, NG2+/PDGFRa+ Plp promoter-expressing progenitors (PPEPs) give rise to neurons in piriform cortex, and provides novel information on both the expression of neurogenic markers by these cortical NG2+/PDGFRa+ PPEPs, and on the phenotypes of the immature and mature neurons derived from this source. Though occasional neurons in other regions of the forebrain (eg, CPu) expressed Plp promoter activity revealed by Cre immunoreactivity (Fig. S3 F
), and hence became EYFP+ early after initiation of tamoxifen injection to PCE/R double transgenic mice (Fig. S3 A-F
), the lack of piriform cortex neuronal Plp promoter expression (Fig. S3 G, H
), and the kinetics of accumulation of EYFP+ neurons in the piriform cortex () were incompatible with the speculation that these EYFP+ neurons were derived from neurons that ectopically express EYFP, since there were virtually no EYFP+ neurons in piriform cortex prior to 17 days post-TM (), at which time-point the recombination rate of OPCs had already reached a plateau level (). While we acknowledge that transgene activity does not necessarily equate to endogenous transcriptional activity, the observations that: (a) all Cre+ cells were Sox10+ in piriform cortex (Fig. S3 I-K
); (b) no Cre+/HuCD+ or Cre+/NeuN+ cells were detected in piriform cortex at any of the time points we assessed (Fig. S3 G-H
); and (c) no EYFP+/NeuN+ () or EYFP+/HuCD+ () were detected shortly after TM treatment, all argue for the fidelity with which the Plp-Cre transgene marked oligodendroglial lineage cells, and not neurons, in adult piriform cortex.
It is very unlikely that SVZ GFAP+ NSCs contributed to this adult cortical neurogenesis, since we did not detect any SVZ GFAP+ cells or DCX+ neuroblasts labeled with EYFP 10 days post-TM treatment of PCE/R mice (), nor did we see EYFP+ piriform cortical neurons in GCE/R mice (Fig. S4 D-E3
). Furthermore, the EYFP+ neurons in adult piriform cortex in our study were glutamatergic pyramidal neurons rather than GABAergic interneurons, which are the most abundant neuronal type known to be derived from SVZ GFAP+ NSCs (Garcia et al., 2004
, Inta et al., 2008
). It has been shown that microglia can specifically fuse to apical dendrites of pyramidal neurons in retrovirus-mediated fate-mapping (Ackman, et al., 2006
). Bearing this in mind, we carefully assessed the EYFP expression in microglia and found no evidence of EYFP+/Iba1+ () or EYFP+/CD11b+ (data not shown) microglia throughout all the time points.
EYFP+ neurons were much less common 60 days post-TM (4 EYFP+/NeuN+ cells out of 1024 EYFP+ cells we counted from 3 mice) in piriform cortex of PCE/R mice in which tamoxifen was first administered to 100 days old PCE/R mice (Fig. S4 A-C
). This result was consistent with the much lower number of EYFP+ OPCs in piriform cortex in these age mice (), again supporting the conclusion that EYFP+ OPCs were the source of piriform cortical EYFP+ neurons.
The existence of adult neurogenesis in cerebral cortex from OPCs has been controversial. Zhu et al. (2008)
reported that no neurons were fate-mapped from NG2+ progenitors in constitutive NG2-Cre mice. Their inability to detect fate-mapped cortical neurons in adult constitutive NG2-Cre mice, despite highly efficient recombination in the oligodendroglial lineage, may have been masked by a background of EYFP+ neurons resulting from ectopic Cre expression in cortex neurons. (see Jackson Laboratory Website, mouse stock number 008533). Fate-mapping in adult Olig2CreERT2
mice also failed to demonstrate labeling of cortical neurons (Dimou et al., 2008
), perhaps because they used older mice and a less robust tamoxifen treatment regimen than we employed in the present study, or alternatively, because the Rosa26-EYFP reporter transgene is a better recombination indicator than Rosa26-EGFP or Z/EG transgenes (Young et al., 2010
). In accord with our own results, fate-mapped piriform projection neurons were detected after intense tamoxifen treatment of young adult PDGFRαCreERT2
mice (Rivers et al., 2008
High level DCX is expressed by migrating and immature neurons in SVZ-RMS-OB and SGZ of DG, two well established adult neurogenic areas (Gage 2000
). DCX is also expressed in other forebrain areas, for example cerebral cortex including neocortex and piriform cortex (Luzzati et al., 2008, Gomez-Climent et al., 2008, Tamura Y et al., 2007
), corpus callosum and striatum (Yang H et al., 2004
), and hypothalamus (Fig. S2 E
). Although some of cortical DCX expressing cells were identified as belonging to neuronal lineages, the identity of a large number of remnant cortical DCX+ cells is unknown (Yang et al., 2004
, Walker et al., 2007
). Here we present evidence that there are two populations of DCX+ cells in piriform cortex. As previously reported (Gomez-Climent et al., 2008), we confirmed that the high level DCX+ cells are neuronal lineage (), whereas the low level DCX-expressing sub-population were positive for PDGFRα () and Sox10 (), designating them as putative OPCs instead of neuronal progenitors (Aguirre and Gallo, 2004
Few EYFP+ neurons incorporated BrdU (Fig. S 4
). This result is similar to the observation by Rivers et al (2008)
mice. There are two possible explanations for the almost complete lack of EYFP+/BrdU+ neurons in adult piriform cortex despite prolonged administration of BrdU in drinking water. First, the EYFP+ neurons may have originated from predominantly post-mitotic EYFP+ OPCs, which comprise ~85% of total EYFP+ OPCs quantified in our study (). This explanation would be consistent with our previous report (Guo et al., 2009
) of substantial numbers of EYFP+/BrdU+ neurons when fate mapping neonatal PCE/R mice, in which a much greater proportion of EYFP+ OPCs are mitotic than in adults. Alternatively, EYFP+ neurons may, in fact, be derived from mitotically cycling OPC, but these cycling adult OPCs, though possible to label by intracerebroventricular administration of BrdU, are not efficiently labeled by systemically administered BrdU (Kokoeva et al., 2005
The piriform cortex we analyzed spans about 1.3 mm from Bregma −2.30 mm to −1.06 mm. Approximately 0.98% of total layer II and III NeuN+ neurons expressed EYFP at 182 days post-TM. By day 182 post-TM, there were approximately 17 EYFP+ neurons per 14 μm section (). About 0.1 EYFP+ neuron was added per 14 μm section of piriform cortex per day during the 180 days after tamoxifen treatment was initiated (17/182 = 0.093). Therefore, approximately 10 [0.1 × (1300/14) = 9.3] FP+ neurons were added to adult piriform cortex daily, which would correspond to about 46 total neurons added [17 ÷ 182 × (1300/14) ÷ 0.2 ≈ 46], if the ~20% recombination rate in total NG2+/PDGFRa+ cells is taken into account. The discrepancy between our calculated result and previous data (24 neurons) (Rivers et al., 2008
) may have been due to the location along the anterior-posterior axis that was evaluated; our data were gathered from posterior piriform cortex, whereas they focused their study on anterior piriform cortex. Alternately, the rate of neuronogenesis from Pip promoter+/PDGFRa+ OPCs is greater than that in the overall pool of PDGFRa+ OPCs.
Our data strongly suggest that OPC-derived neurons became integrated into the neuronal network in adult piriform cortex. What is their functional role? The piriform cortex is not only the first and largest destination for input from the olfactory bulb, but also acts as an association cortex that integrates sensory information from the environment with behavioral, contextual and cognitive input (Haberly 2001
). Given the importance of layer II pyramidal neurons, the role of piriform cortex and the continuous replacement of olfactory bulb interneurons, we hypothesize that the continued addition of OPC-derived neurons to piriform cortex during adulthood is important in preserving the capacity for olfactory discrimination learning and memory, and is driven by continued olfactory bulb input. Consistent with this hypothesis, pyramidal neurons in piriform cortex selectively undergo apoptosis after total bulbectomy (Capurso et al., 1997
). Taken together, our findings prove that PLP promoter-expressing OPCs continue to generate glutamatergic pyramidal neurons in the adult piriform cortex.