Distribution and fate of DCX/PSA-NCAM expressing cells in the adult mammalian cortex: A local reservoir for adult cortical neuroplasticity?

Abstract

The expression of early developmental markers such as doublecortin (DCX) and the polysialylated-neural cell adhesion molecule (PSA-NCAM) has been used to identify immature neurons within canonical neurogenic niches. Additionally, DCX/PSA-NCAM+ immature neurons reside in cortical layer II of the paleocortex and in the paleo- and entorhinal cortex of mice and rats, respectively. These cells are also found in the neocortex of guinea pigs, rabbits, some afrotherian mammals, cats, dogs, non-human primates, and humans. The population of cortical DCX/PSA-NCAM+ immature neurons is generated prenatally as conclusively demonstrated in mice, rats, and guinea pigs. Thus, the majority of these cells do not appear to be the product of adult proliferative events. The immature neurons in cortical layer II are most abundant in the cortices of young individuals, while very few DCX/PSA-NCAM+ cortical neurons can be detected in aged mammals. Maturation of DCX/PSA-NCAM+ cells into glutamatergic and GABAergic neurons has been proposed as an explanation for the age-dependent reduction in their population over time. In this review, we compile the recent information regarding the age-related decrease in the number of cortical DCX/PSA-NCAM+ neurons. We compare the distribution and fates of DCX/PSA-NCAM+ neurons among mammalian species and speculate their impact on cognitive function. To respond to the diversity of adult neurogenesis research produced over the last number of decades, we close this review by discussing the use and precision of the term “adult non-canonical neurogenesis.”

Keywords

aging / cognition / doublecortin / piriform cortex / plasticity / neurogenesis

Neurogenesis in the adult mammalian brain: a controversial topic

The end of a dogma: new neurons are generated in the adult mammalian central nervous system

The subgranular zone of the dentate gyrus and the subventricular zone of the lateral ventricles, defined as the canonical neurogenic niches in the adult mammalian brain, were long believed to be the exclusive sites for the genesis of adult-born neurons. In the healthy rodent brain, adult-born neuroblasts in the subventricular zone migrate tangentially toward the olfactory bulb where they mature into two types of interneurons and integrate into pre-existing networks ( Rosselli-Austin and Altman, 1979; Luskin and Boone, 1994; Betarbet et al., 1996; Zigova et al., 1996; Biebl et al., 2000; Kato et al., 2000; Petreanu and Alvarez-Buylla, 2002; Winner et al., 2002; Carleton et al., 2003; Kelsch et al., 2007). Moreover, adult-born neuroblasts in the subgranular zone of the dentate gyrus differentiate into glutamatergic granular cells and integrate into local circuits ( Brown et al., 2003; Bizon et al., 2004; Bondolfi et al., 2004; Kempermann et al., 2004; Couillard-Despres et al., 2005; Encinas et al., 2011; Vivar and van Praag, 2013). In humans, adult neurogenesis was also reported in these two canonical niches, with noteworthy variability depending on the applied research methods and age of the subjects ( Eriksson et al., 1998; Bédard et al., 2002; Curtis et al., 2007; Manganas et al., 2007; Sanai et al., 2011; Spalding et al., 2013; Ernst et al., 2014). However, recent findings challenge the hypothesis that these canonical neurogenic niches are the only two areas of neurogenesis in the healthy adult brain.

Current question: can adult neurogenesis occur outside the canonical neurogenic niches?

Recent studies suggest the presence of adult-born neurons or immature neurons in several regions of the adult mammalian central nervous system outside the canonical neurogenic niches, including cortical areas ( Kaplan, 1981; Dayer et al., 2005; Takemura, 2005; Shapiro et al., 2007a, 2007b; Shapiro et al., 2009; de la Rosa-Prieto et al., 2010), subcortical areas ( Bédard et al., 2002; Bernier et al., 2002; De Marchis et al., 2004; Vessal et al., 2007; Okuda et al., 2009; Pierce and Xu, 2010; Ehninger et al., 2011; Dirian et al., 2014; Ernst et al., 2014; Luzzati et al., 2014), and the spinal cord ( Shechter et al., 2007). In humans, histological and carbon-14 dating approaches revealed that adult-born interneurons could integrate into the striatum ( Ernst et al., 2014). The presence of adult-born neurons outside the canonical neurogenic niches has been reported in a variety of studies in several mammalian species, whereas their integration has been demonstrated to a lesser extent. Some of these findings could not be replicated in certain mammalian species, while others could not be reproduced since their first publication or have since been contradicted by subsequent studies. It is also possible that some of these findings are based on low-level, “incomplete” neurogenic processes that protracted from embryogenesis into the postnatal period (reviewed in Bonfanti and Peretto, 2011; Feliciano et al., 2015). Despite the controversies, these cortical and subcortical areas are currently classified as the “non-canonical neurogenic regions” of the healthy mammalian brain ( Bonfanti, 2013; Peretto and Bonfanti, 2014; Feliciano et al., 2015). These putative adult neurogenic regions are illustrated in Fig. 1.

Limits and ambiguities of cell-proliferation assays used to research adult neurogenesis

The existence, manner, and origin of adult neurogenesis within non-canonical neurogenic regions have been, and continue to be, controversial topics. Besides species-specific variations ( Bonfanti and Peretto, 2011), a significant limitation arises from the current labeling techniques used to detect neurogenic events. Cell-division markers, such as 5-bromo-2'-deoxyuridine (BrdU) and tritiated thymidine, only label cells in their S-phase ( Nowakowski et al., 1989; Feliciano and Bordey, 2013), resulting in an underestimation of the proliferative cell number because proliferative cells in other phases of their cell cycle are potentially omitted. On the other hand, thymidine analogs can lead to an overestimation of proliferative cell number since these analogs can be assimilated by neurons undergoing DNA repair or non-proliferative DNA synthesis ( Kunz and Kohalmi, 1991; Nowakowski and Hayes, 2000; Yang et al., 2001; Kornack and Rakic, 2001; Kuan et al., 2004; Breunig et al., 2007; Burns et al., 2007). Furthermore, thymidine analogs have been detected in vivo in dead neurons ( Burns et al., 2006), resulting in an overestimation of proliferative cell numbers. Hence, targeting DNA-synthesis to detect neurogenesis results in a failure to detect some adult post-mitotic precursors, and the potential misclassification of labeled cells that have not undergone proliferative events (reviewed in Feliciano and Bordey, 2013). In addition, the side effects of BrdU labeling have been shown to compromise cell proliferation, cell migration, and influence the final cell position and number. BrdU is more toxic than tritiated thymidine, as demonstrated both in vitro in neural stem and progenitor cell cultures ( Lehner et al., 2011) and in vivo in the rhesus monkey ( Duque and Rakic, 2011).

Despite the limitations of conventional DNA-synthesis labeling techniques, if adult neurogenesis were detected independent of cell proliferation, then these labeling techniques would certainly underestimate the neurogenic potential and plasticity of the adult mammalian brain. Notably, it has been recently suggested that post-mitotic cells mature into newly formed neurons in some non-canonical neurogenic regions of the adult mammalian brain ( Gómez-Climent et al., 2008; Luzzati et al., 2009; Gomez-Climent et al., 2010; Bonfanti and Peretto, 2011; Bonfanti and Nacher, 2012; Clarke et al., 2012; Rubio et al., 2015), which raises questions regarding the origin, fate, and functional relevance of these post-mitotic cells in the adult cortex.

How diverse is neurogenesis outside the canonical neurogenic niches?

Evidence for three independent cell types with neuronal fates in the healthy cortex

Adult-born neurons in canonical neurogenic niches derive from neural lineage-restricted proliferating stem and progenitor cells ( Doetsch et al., 1997; Gage et al., 2008). In contrast, there is indirect evidence that even in the healthy nervous system new neurons outside the canonical niches may originate from a variety of different sources, including glial precursor cells. Innovative and meticulous studies have led to the identification of at least three different cell types that give rise to new neurons outside the canonical neurogenic niches of the (healthy) adult mammalian brain. The first was BrdU-positive neuroblasts emigrating from the subventricular zone, which are reported to give rise to a small number of new-born neurons in some ventral brain regions in the early to young postnatal period in mice and rats ( De Marchis et al., 2004; Shapiro et al., 2007a; Shapiro et al., 2007b; Shapiro et al., 2009), as well as in young rabbits and monkeys, where the migratory routes of these immature neurons are well documented ( Bédard et al., 2002; Bernier et al., 2002; Luzzati et al., 2003). However, since this form of neurogenesis was only observed in the very early postnatal period in these mammals, critics stated that this low-level neurogenic process was merely a protraction of an incomplete developmental neurogenesis (reviewed in Bonfanti and Peretto, 2011). Therefore, in this case, the term “adult non-canonical neurogenesis” should be used very carefully.

The second type of putative neuronal precursors is neural/glial antigen 2 (NG2)-expressing cells. NG2-expressing cells are often referred to as oligodendrocyte progenitor cells ( Nishiyama et al., 2009; Nishiyama et al., 2014), or synantocytes ( Butt et al., 2005) and are sometimes, perhaps erroneously, intermixed with NG2-expressing polydendrocytes (own observation). Polydendrocytes are process-bearing glial cells expressing NG2 and the platelet-derived growth factor receptor alpha (PDGFRA). We want to emphasize that while both oligodendrocyte progenitor cells and polydendrocytes express NG2, the function and fate of these cell populations may differ. Evidence suggests that polydendrocytes can give rise to oligodendrocytes under certain conditions (reviewed in Nishiyama et al., 2009), and some astroglial precursors express NG2 during murine corticogenesis ( Zhu et al., 2008). Despite the ambiguity regarding their exact identity, NG2-expressing cells were reported to be present in all cortical areas investigated ( Dawson et al., 2003; Nishiyama et al., 2009; Nishiyama et al., 2014) and can occasionally incorporate BrdU ( Clarke et al., 2012; Psachoulia et al., 2009). In young adult mice, NG2-expressing cells were reported to constitute a local source of new neurons in the piriform cortex, motor cortex, hypothalamus, and amygdala ( Rivers et al., 2008; Guo et al., 2010; Ehninger et al., 2011; Clarke et al., 2012; Robins et al., 2013). However, other studies were unable to validate a possible neurogenic fate of NG2-expressing cells ( Dimou et al., 2008; Komitova et al., 2009; Kang et al., 2010; Zhu et al., 2011). Notably, technical artifacts resulting from transgenic fate mapping models have led to profound misinterpretations regarding the neurogenic potential of NG2-expressing cells in vivo (reviewed in Richardson et al., 2011; Nishiyama et al., 2014).

A third population of cells with a putative neuronal fate is identified by the expression of doublecortin (DCX), polysialylated-neural cell adhesion molecule (PSA-NCAM), or both molecules. These cells reside mostly in cortical layer II of the piriform cortex, the entorhinal cortex, and the neocortex of various mammals ( Bonfanti et al., 1992; Seki and Arai, 1999; Nacher et al., 2001; Nacher et al., 2002; Gómez-Climent et al., 2008; Xiong et al., 2008; Cai et al., 2009; Luzzati et al., 2009; Varea et al., 2009; Varea et al., 2011; He et al., 2014; Yang et al., 2015). Moreover, comparable cells have been found in the amygdala of rats, cats, monkeys, and humans ( Nacher et al., 2002; Martí-Mengual et al., 2013). Cortical cells which co-express both DCX and PSA-NCAM (DCX/PSA-NCAM cells), are likely to constitute a population of immature and post-mitotic neurons in the adult brain, which are not tagged by short-term BrdU pulse-labeling experiments ( Gómez-Climent et al., 2008; Luzzati et al., 2009; Varea et al., 2011; Bonfanti and Nacher, 2012; Rubio et al., 2015; Yang et al., 2015). Nevertheless, these cells maybe a source of new inhibitory ( Xiong et al., 2008; Cai et al., 2009; Zhang et al., 2009) or excitatory neurons ( Luzzati et al., 2009; Varea et al., 2011; Rubio et al., 2015). Until now, the origin, fate, and function of cortical DCX/PSA-NCAM-expressing cells in non-canonical neurogenic niches remain controversial.

The healthy murine piriform cortex might contain all three cell types with neuronal fates

The existence of one cell population with a putative neuronal fate in non-canonical regions does not exclude the presence or relevance of another. Whether or not some pyramidal neurons in the adult murine piriform cortex are derived from non-proliferating NG2-expressing cells ( Guo et al., 2010; Richardson et al., 2011), does not exclude the contribution of subventricular zone-derived neuroblasts to the generation of neuron-specific nuclear protein (NeuN)-expressing cells in very young rodents ( Shapiro et al., 2009; Shapiro et al., 2007a), nor does it exclude that prenatally produced DCX/PSA-NCAM-expressing cells may also be present in cortical layer II of adult mammals ( Cai et al., 2009; Luzzati et al., 2009; Zhang et al., 2009; Varea et al., 2011; Rossi et al., 2014; Rubio et al., 2015). Given previous disagreements, it is possible that these three cell populations, with an assumed neuronal fate, are present in the healthy adult piriform cortex in different quantities according to age and species (e.g. Yuan et al., 2014, 2015 vs. Nacher and Bonfanti, 2015). To further complicate matters, cortical layer I has also been suggested as a fourth source of potential new neurons as an early postnatal, and perhaps young adult, neurogenic niche for cortical GABAergic interneurons under physiological conditions ( Costa et al., 2007; Xiong et al., 2010). The diversity of cell populations suspected to generate or to differentiate into neurons in the piriform cortex allows this primal three-layered cortex to be one of the most suitable and interesting brain regions to study non-canonical neurogenesis in the adult mammalian brain. Deciphering origins and fates of different neuronal and non-neuronal, migrating and non-migrating, proliferating and non-proliferating progenitors or residing immature cells with delayed maturation, may lead to the elucidation and classification of the basic forms of adult cortical neurogenesis in lower and higher mammals.

The remainder of this review will focus on the alleged form of proliferation-independent maturation of neurons from cortical DCX/PSA-NCAM-expressing cells. We discuss the age-related decrease in DCX/PSA-NCAM-expressing cells, examine their distribution and plausible fates in various mammalian species, and consider their possible physiologic role in cortical circuitry. We will conclude this review by discussing the usage and precision of the term “adult non-canonical neurogenesis.”

The co-expression of DCX and PSA-NCAM defines immature neurons in the adult brain

DCX and PSA-NCAM are markers of neuronal development and plasticity

The microtubule-associated protein DCX and the cell surface protein PSA-NCAM are molecules related to neuronal development and cellular plasticity ( des Portes et al., 1998; Francis et al., 1999; Seki and Arai, 1999; Couillard-Despres et al., 2005; Friocourt et al., 2007). DCX is associated with dynamic instability and in turn, is crucial for axonal elongation and dendritic sprouting ( Hastings and Gould, 1999; Ehninger and Kempermann, 2008). The polysialylated (PSA) form of the neuronal cell adhesion molecule (NCAM)can produce a sufficient physical hindrance between apposing membranes to attenuate intercellular adhesion ( Johnson et al., 2005; Rutishauser, 2008). Thus, PSA-NCAM is involved in synaptogenesis and activity-dependent remodeling of synapses and is frequently used to identify migrating neuroblasts ( Dityatev et al., 2004; Saegusa et al., 2004). Both DCX and PSA-NCAM are predominantly co-expressed in immature neurons during development. In adult mammals, the co-expression of DCX and PSA-NCAM persists in the canonical neurogenic niches and reflects neurogenic activity ( Gritti et al., 2002; Couillard-Despres et al., 2005; Gould, 2007). However, the presence of either DCX or PSA-NCAM does not solely define immature neurons, since these proteins can also be detected in non-neurogenic, highly plastic cells in the adult mammalian brain ( Fox et al., 2000; Nacher et al., 2001; Sairanen et al., 2007; Varea et al., 2007; Kremer et al., 2013). Therefore, in the adult brain, the co-expression of DCX and PSA-NCAM is frequently used to detect immature neurons, while the individual expression of either DCX or PSA-NCAM is used to categorize maturation stages or to indicate plasticity in mature neural cells.

Most cortical DCX/PSA-NCAM-expressing cells are immature neurons

Several lines of evidences indicate that DCX/PSA-NCAM expressing cells within the mammalian paleocortex, entorhinal cortex, and neocortex are immature neurons committed to a neuronal fate. The first evidence is that most of these cells are negative for markers of mature principal neurons and interneurons ( Gómez-Climent et al., 2008; Cai et al., 2009; Luzzati et al., 2009; Varea et al., 2011; Rubio et al., 2015). Second, they are devoid of ultrastructural features that indicate synaptic inputs, as shown by transmission electron microscopy ( Gómez-Climent et al., 2008). In addition, electron microscopic analysis by Shapiro and colleagueś (2007) revealed that DCX labeled cells had ultrastructural features of immature migrating cells. Third, DCX/PSA-NCAM-expressing cells are negative for mature oligodendroglial, astroglial and microglial markers ( Gómez-Climent et al., 2008; Luzzati et al., 2009; Rubio et al., 2015), and very few DCX/PSA-NCAM-expressing cells express NG2 within the piriform cortex ( Gómez-Climent et al., 2008; Luzzati et al., 2009; Rubio et al., 2015). Fourth, the expression of DCX/PSA-NCAM in cortical cells overlaps with other early developmental markers typically found in immature neurons during development, such as TUC-4, TUJ-1, and CNGA3 ( Gómez-Climent et al., 2008; Xiong et al., 2008; Luzzati et al., 2009). Finally, DCX/PSA-NCAM-expressing cells do not co-express cell activity markers, such as c-Fos or Arc, further emphasizing their immature state ( Gómez-Climent et al., 2008). In sum, DCX/PSA-NCAM-expressing cells, at least in the piriform cortex, are considered as bona fide latent immature neurons in cortical layer II and a potential reservoir of new mature neurons upon differentiation. For this reason, we have named these cells, “cortical layer II DCX/PSA-NCAM-expressing immature neurons,” abbreviated as “DCX/PSA-NCAM IN.” Aside from immunohistochemistry, these cells can be identified in transgenic mice overexpressing fluorescent proteins under the control of a human DCX promoter ( Couillard-Despres et al., 2006) or in genetic fate mapping in transgenic mice expressing CreER^T2 under the control of the same promoter ( Zhang et al., 2010). The distribution of immature neurons targeted by the DCX promoter is shown in Fig. 2. Most DCX promoter positive cells in layer II of the murine piriform cortex co-express PSA-NCAM (own observation). It is worth mentioning that a minority of DCX/PSA-NCAM-expressing cells with morphologies comparable to semilunar-pyramidal transitional neurons also express NeuN ( Gómez-Climent et al., 2008; Varea et al., 2011; Rubio et al., 2015). Ultrastructural analysis revealed some synaptic inputs to the distal dendrites of larger DCX/PSA-NCAM/NeuN-expressing semilunar-pyramidal transitional neurons ( Gómez-Climent et al., 2008).

Origin, anatomical distribution, and fate of cortical layer II DCX/PSA-NCAM-expressing immature neurons (DCX/PSA-NCAM IN)

DCX/PSA-NCAM IN are post-mitotic

DCX/PSA-NCAM IN do not appear to be the product of cell division in adulthood. Instead, most of these cells arise in the piriform cortex during embryonic development: between E13.5 and E14.5 in mice according to BrdU incorporation experiments ( Rubio et al., 2015), between E13.5 and E15.5 in rats ( Gómez-Climent et al., 2008), between E21 and E28 in guinea pigs, and within the neocortex of guinea pigs at E35 ( Yang et al., 2015). Some reports have described marginal BrdU incorporation in DCX/PSA-NCAM IN during adulthood ( Shapiro et al., 2007a). However, the vast majority of DCX/PSA-NCAM IN observed in layer II of the piriform cortex and entorhinal cortex of adult mice and rats, as well as the piriform cortex, entorhinal cortex, and neocortex of guinea pigs, rabbits, and cats are post-mitotic immature resident neurons ( Gómez-Climent et al., 2008; Luzzati et al., 2009; Klempin et al., 2011; Varea et al., 2011; Rubio et al., 2015). In conclusion, these studies unanimously indicate that the majority of layer II DCX/PSA-NCAM IN do not replicate their DNA in early or late adulthood, and thus, are not adult-born.

Further studies, which did not use proliferation markers, suggested that outside canonical neurogenic regions the DCX/PSA-NCAM IN are most abundant in layer II of the piriform cortex, entorhinal cortex, and neocortex of four afrotherian mammals ( Patzke et al., 2014), young lupines ( De Nevi et al., 2013), non-human primates ( Cai et al., 2009; Zhang et al., 2009; Bloch et al., 2011), and humans ( Cai et al., 2009; Mikkonen et al., 1998; Ni Dhúill et al., 1999). Although some uncertainty about the exact identity of DCX/PSA-NCAM IN in these studies resulted from the absence of BrdU administration and the variability associated with post-mortem histological analyses, the distribution and morphology of cortical cells expressing DCX and/or PSA-NCAM seemed to be comparable between these mammalian species. Therefore, we speculate that cortical DCX/PSA-NCAM IN are generated at prenatal stages not only in mice, rats, and guinea pigs, but also in other mammals, including cats, lupines, and primates. We propose that cortical DCX/PSA-NCAM IN constitute a population of resident immature neurons that are not adult-born (reviewed in Gómez-Climent et al., 2008; Nacher and Bonfanti, 2015).

The number of DCX/PSA-NCAM IN declines drastically with age

An age-dependent decrease in the number of DCX/PSA-NCAM IN was observed in the cortical areas of mice, rats, guinea pigs, rabbits, cats, dogs, and non-human primates ( Abrous et al., 1997; Murphy et al., 2001; Xiong et al., 2008; Cai et al., 2009; Varea et al., 2009; Zhang et al., 2009; De Nevi et al., 2013). In cortical layer II, such a decrease could results from cell death, migration, differentiation, or maturation into adult neuronal cells. Current studies have failed to find evidence of elevated TUNEL activity in cortical layer II during aging in guinea pigs ( Xiong et al., 2008) or a substantial number of pyknotic nuclei in aged rats (Gomez-Climent and Nacher, unpublished results). Maturation would imply a loss of developmental markers (DCX/PSA-NCAM) in immature cortical neurons and the progressive appearance of mature neural cell markers. Therefore, if DCX/PSA-NCAM IN constitute an immature cell population, upon maturation, these cells would be untraceable in aged mammalian brains and indistinguishable from their neighboring mature neurons. Transgenic labeling is an effective strategy to follow the fate of immature neurons and neuronal progenitor cells, even when the expression of markers such as DCX and PSA-NCAM declines. An example of fate-mapping analysis to address the maturation hypothesis of DCX/PSA-NCAM IN using the DCX-CreER^T2 mouse line is shown in Fig. 3 (own observation).

The “loss by maturation” hypothesis: DCX/PSA-NCAM IN mature and integrate during adulthood

Previous observations have supported the “loss by maturation” hypothesis. The first observation was that some cortical PSA-NCAM-expressing cells, with bigger somata than DCX/PSA-NCAM co-expressing cells, were endowed with synaptic spine-like protrusions and thin axon-like basal processes. These morphologies typically identify semilunar-pyramidal transitional neurons in the piriform cortex of mice, rats, and cats ( Gómez-Climent et al., 2008; Varea et al., 2011; Rubio et al., 2015). Furthermore, these larger PSA-NCAM-expressing cells faintly co-expressed NeuN and the NMDA-receptor subunit NR1 in the piriform cortex of mice and the piriform cortex and entorhinal cortex of rats ( Gómez-Climent et al., 2008; Rubio et al., 2015). Therefore, these cells may represent the final path or intermediate state of DCX/PSA-NCAM IN toward maturation. Due to their location, others have suggested that DCX/PSA-NCAM IN mature into semilunar, pyramidal, or semilunar-pyramidal transitional neurons in the piriform cortex. In the piriform cortex, most excitatory neurons are clustered in layer II, while GABAergic interneurons are sparsely spread across all layers, as shown in Fig. 3 ( Kapur et al., 1997; Ekstrand et al., 2001; Suzuki and Bekkers, 2007, 2010a, 2010b). Among excitatory neurons, semilunar cells are particularly concentrated in the outer part of layer II, where most DCX/PSA-NCAM IN can be found in the piriform cortex of mice and rats ( Gómez-Climent et al., 2008; Rubio et al., 2015). Thus, it has been suggested that semilunar cells may be the result of the maturation of DCX/PSA-NCAM IN, at least in the piriform cortex ( Gómez-Climent et al., 2008; Luzzati et al., 2009; Gomez-Climent et al., 2010). While these observations support the “loss by maturation” hypothesis, the fate of DCX/PSA-NCAM IN to become either excitatory neurons and/or inhibitory neurons is still unknown.

The fate of DCX/PSA-NCAM IN: glutamatergic or GABAergic?

The fate of DCX/PSA-NCAM IN remains elusive. One laboratoryhas suggested that DCX/PSA-NCAM IN mature into inhibitory neurons in guinea pigs, cats, and primates ( Xiong et al., 2008; Cai et al., 2009; Zhang et al., 2009). Others have substantiated that DCX/PSA-NCAM IN provide a post-mitotic pool of excitatory neurons in mice, rats, guinea pigs, rabbits, and cats ( Gómez-Climent et al., 2008; Luzzati et al., 2009; Varea et al., 2011; Rubio et al., 2015). A detailed summary of these reports are given in Table 1, which highlights the discrepancies in expected fates, while also showing the species, locations, birth-dating methods, morphologies, and applied markers of each study. The transcription factor protein Tbr1, a marker of glutamatergic differentiation in pallium-derived neurons, has been used in studies reporting an excitatory fate for DCX/PSA-NCAM IN ( Englund et al., 2005; Hevner et al., 2006). Luzzati and colleagues (2009) observed in mice, rats, guinea pigs, and rabbits that almost all DCX-expressing cortical neurons with immature morphology were also positive for Tbr1, while only a minority of post-mitotic DCX-expressing cells could be co-labeled with interneuron markers. Similarly, Rubio and colleagues (2015) found DCX/PSA-NCAM IN to be positive for Tbr1 in the paleocortical layer II of mice. Moreover, a recent study reported that small paleo-, entorhinal-, and neocortical PSA-NCAM-expressing cells seem to follow a predominantly glutamatergic fate, with virtually no overlap between PSA-NCAM and inhibitory neuronal markers in the cortices of cats ( Varea et al., 2011). Some large DCX/PSA-NCAM-expressing cells co-express NeuN and have morphologies similar to common excitatory morphotypes, such as the semilunar-pyramidal transitional neurons ( Gómez-Climent et al., 2008; Varea et al., 2011; Rubio et al., 2015), indicating a maturation of DCX/PSA-NCAM IN into glutamatergic cell types. In summation, of the five species examined, an excitatory fate for cortical DCX/PSA-NCAM IN is assumed in all three studies. Notably, four out of the five species are small mammals, and all researchers administered BrdU and restricted their quantitative analysis to post-mitotic immature neurons in paleocortical areas, or in the entorhinal cortex.

Other studies suggest an inhibitory neuronal fate for cortical DCX/PSA-NCAM IN. These conflicting observations are all based on studies from a single laboratory, which have found co-expression of DCX (but not PSA-NCAM) and GABAergic markers in larger mammals, such as cats, and non-human primates ( Cai et al., 2009; Zhang et al., 2009), or relied on slightly ambiguous morphological criteria in dogs and humans ( Cai et al., 2009; De Nevi et al., 2013). In non-human primates, Zhang and colleagues (2009) found a complete co-localization of DCX and PSA-NCAM among small cells and a co-localization NeuN or GABA with low levels of DCX (but not PSA-NCAM) in larger cortical layer II cells. These results directly contrast those of Varea et al., (2011), who observed that small unipolar and bipolar DCX-expressing cells did not exhibit NeuN or GABA reactivity in the entorhinal cortex of cats, but that larger cells with complex morphology did co-expressed DCX, NeuN, and GABA ( Varea et al., 2011). In guinea pigs, an almost complete co-localization of DCX,PSA-NCAM, and Tuj-1 was found in cortical layer II cells with immature morphology, while larger cells co-expressed DCX, NeuN, and different interneuron markers ( Xiong et al., 2008). Inconsistencies among these studies are likely the result of technical issues as well as species specific and regional variation in the distribution and fate of cortical DCX/PSA-NCAM IN. Since neither Xiong and colleagues (2008) nor Cai and colleagues (2009) used proliferation-markers, an intermixture of post-mitotic cortical layer II DCX/PSA-NCAM IN with proliferating precursors from cortical layer I or the subventricular zone could account for some of the reported discrepancies regarding the fate of strictly post-mitotic DCX/PSA-NCAM IN. In addition, species-specific variations in the distribution and fate of DCX/PSA-NCAM IN may reflect phylogenetic aspects of corticogenesis and cortical processing, as schematically represented in Box 1.

Before a clear line of evidence can rule out or support the hypothesis of species-specific distribution/fate/population of DCX/PSA-NCAM IN, four systematic problems have to be overcome. First, none of the studies reporting or suggesting maturation toward inhibitory neurons administered proliferation markers ( Xiong et al., 2008; Cai et al., 2009; Zhang et al., 2009; De Nevi et al., 2013). Thus, studies suggesting a GABAergic fate of DCX/PSA-NCAM IN may include mitotic DCX expressing cortical cells in their quantitative analysis, as well as migrating neuroblasts of the subventricular zone, which are BrdU positive, and local proliferating progenitors. Secondly, fixation and staining protocols need to be standardized since they currently vary among the different studies and species investigated. Thirdly, the molecular marker used to follow the fate of DCX/PSA-NCAM IN is DCX in some studies ( Xiong et al., 2008; Cai et al., 2009; Zhang et al., 2009; De Nevi et al., 2013; Patzke et al., 2014; Yang et al., 2015;), but in others is PSA-NCAM ( Varea et al., 2009; Varea et al., 2011). To follow the fate of DCX/PSA-NCAM IN non-ambiguously, we recommend the use of both antibodies simultaneously in combination with Tbr1 ( Luzzati et al., 2009; Rubio et al., 2015). We also suggest the need for transgenic mouse models that can be utilized in proof of principle studies. Finally, to compare the fate of DCX/PSA-NCAM IN between species, it would be essential to investigate and compare the same regions across all species, e.g. allocortical areas, since the fate of DCX/PSA-NCAM IN may be region specific. For instance, small mammals, such as rodents, do not have comparable neocortical lobes to those observed in cats, dogs, and primates, and thus, the lack of neocortical DCX/PSA-NCAM IN in small mammals could bias the comparison when DCX/PSA-NCAM IN are considered across all species. Regardless of whether DCX/PSA-NCAM IN develop into glutamatergic or GABAergic neurons, the available studies, once more, unanimously suggest that recently formed neurons during adulthood arise from the maturation of layer II immature neurons.

The relevance of DCX/PSA-NCAM IN in adult cortices: facts, speculations, and definitions

DCX/PSA-NCAM IN distribution parallels cortical hierarchy: functional implications

DCX/PSA-NCAM IN are more abundant in secondary, or associative, cortical regions than in primary sensory areas. The motor cortex in most investigated species is almost completely devoid of DCX/PSA-NCAM IN ( Gómez-Climent et al., 2008; Luzzati et al., 2009; Zhang et al., 2009; Rubio et al., 2015). Therefore, the distribution of DCX/PSA-NCAM IN may parallel the hierarchical levels of sensory processing. In the young adult monkey, DCX/PSA-NCAM IN are widely distributed across the entire cerebral hemisphere, denser in layer II/III with a decreasing ventrodorsal gradient ( Cai et al., 2009; Zhang et al., 2009). In the neocortex of guinea pigs and rabbits, the number of DCX-expressing cells increases from primary to secondary sensory areas, reaching the highest density close to the rhinal sulcus, which marks the border between the neocortex and the limbic system ( Luzzati et al., 2009). Around the rhinal sulcus, layer II DCX-expressing cells can also be found in the mouse and rat ( Nacher et al., 2001; Gómez-Climent et al., 2008; Luzzati et al., 2009). However, in these two species DCX/PSA-NCAM IN are predominantly located in the posterior part of the piriform cortex ( Gómez-Climent et al., 2008; Luzzati et al., 2009; Rubio et al., 2015). This brain region is an associative cortical area, thought to be responsible for higher-order olfactory processing ( Neville and Haberly, 2003; Gottfried et al., 2006; Kadohisa and Wilson, 2006a, 2016b; Bekkers and Suzuki, 2013). Hence, the recurrent observation among different species is that DCX/PSA-NCAM IN are most abundant in higher-order integrative cortical areas, suggesting that these cells play strategic roles in advanced brain functions.

DCX/PSA-NCAM IN might constitute a consumable reservoir for cortical plasticity crucial for cognition

Similar to the adult-born neurons in the dentate gyrus of young rodents, which are thought to promote plasticity of hippocampal circuits and therefore cognitive function (( Gould et al., 1999; Ambrogini et al., 2000; Lemaire et al., 2000; Shors et al., 2001; Shors et al., 2002; Bizon and Gallagher, 2005), reviewed in König et al., 2016), the integration of cortical DCX/PSA-NCAM IN is predicted to provide a form of cellular plasticity for higher-order cortical circuits. The integration of neuronal precursors into existing hippocampal networks ( Cameron and McKay, 1999; Hastings and Gould, 1999; Markakis and Gage, 1999; van Praag et al., 2002) leads to competition with pre-established connections ( Ge et al., 2006; Toni et al., 2008; Toni et al., 2007), and provides an alternative method by which synaptic plasticity can occur ( Gould et al., 1999; Toni et al., 2008; Toni et al., 2007). Similarly, maturation and integration of DCX/PSA-NCAM IN in the associative cortices and higher-order (secondary) sensory areas may facilitate remodeling and fine-tuning capacity of pre-existing cortical networks in adult mammals. Interestingly, the cortical areas containing the highest density of DCX/PSA-NCAM IN are commonly reported to be crucial for the integration of different modalities, including attention, planning, and memory storing ( Purves et al., 2001). Accordingly, we propose that DCX/PSA-NCAM IN constitute a limited, post-mitotic, local reservoir for cortical plasticity, and as such, a consumable source for cognitive plasticity during aging.

DCX/PSA-NCAM IN and the term “adult non-canonical neurogenesis”

Could the delayed maturation and integration of DCX/PSA-NCAM IN outside neurogenic niches be considered adult non-canonical neurogenesis? In the cortex, DCX/PSA-NCAM IN are generated prenatally and undergo late maturation. Thus, they are newlymatured, functional neurons but not newly born cells. To help determine how this peculiar form of cellular development can be classified as non-proliferative/non-canonical neurogenesis, we will briefly discuss the different types of mammalian neurogenesis. The first type is developmental neurogenesis: the formation of functionally mature neurons from proliferating neural stem cells during embryonic development, which can be protracted into early postnatal and adult ages in a species-specific manner ( Bonfanti, 2013). The second type is adult neurogenesis: the formation of functionally mature neurons from proliferating neural stem or progenitor cells in adult canonical neurogenic niches and possibly other yet to be defined brain areas. We propose a third type of neurogenesis, which is a hybrid between developmental and adult neurogenesis. Here, cortical cells are born during embryonic development, but prematurely interrupt their maturation and can be identified as DCX/PSA-NCAM IN in the adult brain. Eventually, these cells can become newlymatured, functional neurons during adulthood. Therefore, in the beginning, DCX/PSA-NCAM IN follow normal patterns of developmental and proliferative, reflecting an incomplete neurogenesis. Later, the maturation of DCX/PSA-NCAM IN into functional neurons that integrate into pre-established networks resembles the process of adult neurogenesis without proliferation. In conclusion, this form of neurogenic or neurogenic-like process could be defined as “suspended developmental neurogenesis” or “adult non-proliferative neurogenesis.” Since the neurogenesis of DCX/PSA-NCAM IN is better characterized by their adult maturation rather than their perinatal undifferentiated proliferation, the second definition is arguably the most accurate when summarizing this two-step process.

Nevertheless, we conclude that the impact of newlymatured, functional neurons on cortical networks may equal the plasticity provided byother adult-born neurons, regardless of the applied terminology. We emphasize the need for further research to validate the “loss by maturation” hypothesis using transgenic animals. Understanding the physiologic potential of DCX/PSA-NCAM IN might offer new insights into brain aging and plasticity during adulthood.

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