Old cells in new layers: the strange case of the DCX+ cells in the layer II of different amniote pallial derivatives
Doublecortin (DCX) is a microtubule associated protein involved in cytoskeletal dynamics during migration and differentiation of immature neurons (Francis et al., 1999; Gleeson et al., 1999; Friocourt, 2003). Accordingly, in the adult brain the expression of DCX is restricted to regions of ongoing neurogenesis (Nacher et al., 2001; Brown et al., 2003; Couillard-Despres et al., 2005; Luzzati et al., 2006; Balthazart and Ball, 2014). The only clear exception to this rule is a population of neurons in the layer II of the piriform cortex and neocortex (Gómez-Climent et al., 2008; Luzzati et al., 2009) that are not adult generated but show a strong and homogeneous DCX immunoreactivity that closely resembles that of immature neurons. Layer II DCX+ cells occurs in two main morphological subtypes: Type I cells have small cell bodies and dendrites confined to layer II, while type II cells have larger cell bodies and send one or two dendritic branches to layer I (Luzzati et al., 2009). Electrophysiological analyses in DCX-GFP mice piriform cortex revealed that type I cells resemble immature neurons, while most type II cells shows mature features with large Na+ currents and multiple action potentials (Klempin et al., 2011). In both piriform cortex and neocortex type I and II DCX+ cells express Tbr1 suggesting that they are glutamatergic neurons derived from pallial germinative zones (Englund et al., 2005; Hevner et al., 2006; Luzzati et al., 2009). Interestingly the clear predominance of subpial dendrites over basal dendrites place type II cells within the population of atypical pyramidal cells previously defined as “extraverted neurons” (Sanides and Sanides, 1972). Since the lack of basal dendrites represent an ancient feature in the evolution of pyramidal cells, extraverted neurons in the neocortex were originally considered a conserved cell type. Besides laboratory mice and rats (Nacher et al., 2001; Luzzati et al., 2009), in which layer II DCX+ cells are scarce and mostly restricted to the piriform and perirhinal cortices (Nacher et al., 2001), in all other mammalian species analyzed so far such as rabbits (Luzzati et al., 2009), guinea pigs (Xiong et al., 2008; Luzzati et al., 2009), cats (Cai et al., 2009), dogs (De Nevi et al., 2013), giant african mole rats (Olude et al., 2014), epaulatted fruit bats (Gatome et al., 2010), reshus macaques (Cai et al., 2009; Fung et al., 2011), and humans (Cai et al., 2009), DCX+ cells in layer II are abundant and widely distributed in both piriform cortex and neocortex. A detailed analysis of the distribution of these cells in rabbits and guinea pigs revealed that layer II DCX+ cells are specifically associated to the network of brain regions connected to the lateral entorinal cortex (LEC; Figure 2A; Luzzati et al., 2009). These brain regions, including the rostro-lateral neocortex and piriform cortex, receive information about local sensory objects and have been implicated in non-spatial cognition. By contrast caudo-medial neocortical areas connected to the Medial EC (MEC) and processing information of both external and internal stimuli involved in spatial cognition, are mostly negative for DCX (for anatomical and functional descriptions of LEC and MEC connections see Burwell and Amaral, 1998a,b; Jones and Witter, 2007; Knierim et al., 2014). Within LEC connected networks the DCX+ cells show a strong preferential distribution in higher order areas such as posterior piriform cortex, secondary sensory areas, insular, perirhinal cortex and prefrontal cortex (Figure 2A). Altogether, the similarities in the morphology, laminar position and preferential distribution in higher order areas strongly suggests that DCX+ cells of the neocortex and piriform cortex may represent a common cell type that is shared by these two regions.
Figure 2  Distribution of DCX+/Tbr1+cells in the pallium of mammals and a lizard. (A) The distribution of layer II DCX+/Tbr1+ cells either newly generated (yellow) or non-newly generated (red) is shown in a schematic view of mammalian allocortical and neocortical regions. The neocortex is schematized according to Sanides in three concentric rings: peri-allocortex, proisocortex, and isocortex. The isocortex contains the primary sensory areas from which information flows through a hierarchichal sequence of areas and reaches either the lateral (LEC) or medial enthorinal cortex (MEC). Areas connected with LEC and MEC are shown in pink and turquoise green respectively, feedforward (solid arrows) and feedback (dashed arrow) pathways are also shown. (B) Schematic coronal sections showing the distribution of DCX+/Tbr1+ cells in different pallial domains in mammals (left) and lizard (right). Additional abbreviations: A1, primary auditory cortex; AC, anterior cingulate cortex; DP, dorsal pallium; IL, infralimbic cortex; Ins, insular cortex; pPC posterior piriform cortex; aPC; anterior piriform cortex; PreL, prelimbic cortex; PreS, presubiculum; POR, postrhinal cortex; Prh, perirhinal cortex; RSP, retrosplenial cortex; SUB, subiculum. Redrawn from Luzzati et al. (2009). Notably, in the lizard L. Muralis we identified DCX+/Tbr1+ cells morphologically similar to those of mammals in the layer II of the olfactory cortex and DVR, with a preferential distribution in higher order areas, but not in the dorsal cortex (Figure 2B). When compared to the DCX+/Tbr1+ cells in the neocortex, the general distribution of these cells in the lizard was consistent with the homologies proposed by Karten (Karten, 1969; Butler et al., 2011). Indeed, the DVR has been proposed to be homologous to temporal neocortical areas, such as auditory and secondary somatosensory and visual cortices, that in mammals show high numbers of DCX+/Tbr1+ cells. By contrast, the neocortical regions proposed as homologous of the dorsal cortex, that include primary somatosensory and visual cortices as well as the posterior cingulate, retrosplenial, and postrhinal cortices, are largely devoid of DCX+/Tbr1+ cells. Collectively, these data strongly support that layer II DCX+/Tbr1+ cells represent a conserved cell type in amniotes. In addition, although the sauropsids homologs of mammalian MEC and LEC associated circuits are still poorly defined (Rattenborg and Martinez-Gonzalez, 2011; Allen and Fortin, 2013; Abellán et al., 2014), it is tempting to speculate that non-newly generated DCX+/Tbr1+ cells may be involved in a conserved form of structural plasticity selectively associated to higher order areas of non-spatial learning and memory networks in amniotes. At the same time, our data suggests that the presence of DCX+/Tbr1+ cells in DP derivatives may represent a mammalian innovation. This supports the hypothesis of Reiner that the UL are an evolutionary novelty, but in parallel introduces the possibility that this novelty has been produced by re-using (or co-opting) pre-existing cell types. In particular, we propose that in the transition from the stem-amniote to mammals, DP progenitors instead of exiting from the cell cycle after the production of the DL neurons homologs, continued to proliferate by setting up a LP and/or VP developmental program, giving rise to the UL of the NC. Thus, the evolution of the neocortex could be attributed to a spatial to temporal patterning switch involving DP and LP/VP developmental programs. An interesting aspect of this model is that it could reconcile the developmental data supporting the field homology of the primary progenitors, with the striking similarities existing between neurons of the neocortex and LP and VP derivatives of sauropsids. Future studies in other reptilian species will be required to understand if the distribution of DCX+/Tbr1+ cells in L. Muralis represents the basal reptilian condition or, as happen in mice, this species simply lack this feature. An important point will be also to define where and when these cells are generated in different tetrapod species. Indeed, previous studies have shown that the VP and LP progenitors give rise to neurons that tangentially migrate to the neocortex in mice (Puelles, 2011; Teissier et al., 2012). Most of these VP/LP derived cells have a transient existence in mice, but we cannot exclude that in other mammalian species some of these cells may persist for longer post-natal periods (Teissier et al., 2010, 2012). Finally, molecular and functional analyses will be necessary to understand if these cells in different amniote species and pallial derivatives actually represent sister cell types. Nonetheless, as we will discuss in the next paragraphs, beside this intriguing cell population the hypothesis of the co-option of the LP/VP developmental program is supported also by other anatomical and developmental data.