Doublecortin expression in adult cat and primate cerebral cortex relates to immature neurons that develop into GABAergic subgroups

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Abstract

DCX-immunoreactive (DCX+) cells occur in the piriform cortex in adult mice and rats, but also in the neocortex in adult guinea pigs and rabbits. Here we describe these cells in adult domestic cats and primates. In cats and rhesus monkeys, DCX+ cells existed across the allo- and neocortex, with an overall ventrodorsal high to low gradient at a given frontal plane. Labeled cells formed a cellular band in layers II and upper III, exhibiting dramatic differences in somal size (5–20 μm), shape (unipolar, bipolar, multipolar and irregular), neuritic complexity and labeling intensity. Cell clusters were also seen in this band, and those in the entorhinal cortex extended into deeper layers as chain-like structures. Densitometry revealed a parallel decline of the cells across regions with age in cats. Besides the cellular band, medium-sized cells with weak DCX reactivity resided sparsely in other layers. Throughout the cortex, virtually all DCX+ cells co-expressed polysialylated neural cell adhesion molecule. Medium to large mature-looking DCX+ cells frequently colocalized with neuron-specific nuclear protein and γ-aminobutyric acid (GABA), and those with a reduced DCX expression also partially co-labeled for glutamic acid decarboxylase, parvalbumin, calbindin, β-nicotinamide adenine dinucleotide phosphate diaphorase and neuronal nitric oxide synthase. Similar to cats and monkeys, small and larger DCX+ cells were detected in surgically removed human frontal and temporal cortices. These data suggest that immature neurons persist into adulthood in many cortical areas in cats and primates, and that these cells appear to undergo development and differentiation to become functional subgroups of GABAergic interneurons.

Introduction

Doublecortin (DCX) is a microtubule-associated protein critical for radial and tangential migration of immature neurons in the developing brain (des Portes et al., 1998, Gleeson et al., 1999, Francis et al., 1999, Friocourt et al., 2007). Persistent DCX expression occurs in discrete adult brain regions (Nacher et al., 2001). This adult expression in the subgranular and subventricular zones reflects neurogenic activity giving rise to new granule cells in the hippocampal dentate gyrus and interneurons in the olfactory bulb (Gritti et al., 2002, Couillard-Despres et al., 2005, Gould, 2007). In general, DCX expression in developing neurons declines with cell differentiation and maturation. Therefore, a partial colocalization of DCX with some terminal neuronal markers, e.g., neuron-specific nuclear protein (NeuN), can be detected among a cohort of immature neurons undergoing development (Brown et al., 2003).

Cells concurrently expressing DCX and other immature neuronal markers, e.g., polysialylated neural cell adhesion molecule (PSA-NCAM) and neuron-specific tubulin-III (TuJ1), are reported in layers II/III in mouse/rat piriform cortex (Seki and Arai, 1991, Bonfanti et al., 1992, Nacher et al., 2001), and in primate entorhinal/temporal cortex (Bernier et al., 2002, Tonchev et al., 2003). PSA-NCAM+ cells are also described in rat and human medial prefrontal cortex (Fox et al., 2000, Sairanen et al., 2007, Varea et al., 2007). These cortical cells were classified as mature neurons undergoing structural plasticity (Nacher et al., 2001), and have been lately clarified as immature neurons (Gómez-Climent et al., 2008). Controversy remains as to whether they are born prenatally (Gómez-Climent et al., 2008) or postnatally (Bernier et al., 2002, Pekcec et al., 2006, Shapiro et al., 2007, Shapiro et al., 2008).

Unlike mice and rats, DCX+ cells in adult guinea pigs and rabbits reside in layer II throughout the neocortex in addition to allocortex (Xiong et al., 2008, Luzzati et al., 2008). Therefore, DCX+ cells might have a wide neocortical distribution in higher mammalian species. To explore this possibility, the present study determined DCX+ cells in adult domestic cat, rhesus monkey and human cerebral cortex. DCX+ cells resembling immature and developing neurons were detected in broad cortical areas in these species, with small cells occurring in layers II and upper III and larger ones in all layers. Almost all DCX+ cells co-expressed PSA-NCAM. Relatively larger DCX+ cells with mature-looking morphology colocalized with NeuN, and in part with several terminal markers of interneurons, including γ-aminobutyric acid (GABA), glutamic acid decarboxylase (GAD67), parvalbumin (PV), calbindin (CB), and β-nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) or neuronal isoform of nitric oxide synthase (nNOS). Collectively, DCX+ cells likely represent a wave of immature and maturing neurons undergoing development and differentiation into presumably GABAergic subpopulations. Thus, interneuron development may extend beyond puberty to adulthood in many parts of the cerebral cortex in high mammalian species, and this protracted course of interneuron development may be essential for cortical morphogenesis, plasticity and functionality.

Section snippets

Animal and tissue preparation

Domestic cats aged at 15, 17, and 21 months [young adults, male, mean = 1.5 ± 0.3 year-old (yr-old)] and 42, 55, 59 and 62 months (adults, male, mean = 4.5 ± 0.7 yr-old), weighing 2.1–4.3 kg, were obtained from the animal facility of Hunan Agricultural University (Changsha, Hunan, China). The brains were used to characterize cortical DCX+ cells including a potential age-related decline of the cells in this species (Xiong et al., 2008). Animals were perfused via the ascending aorta with 4%

Distribution of DCX+ cells in young adult cat cortex

In the frontal lobe sections, DCX+ cells and processes were most dense in the subventricular zone lining the anterior horn of the lateral ventricle (Figs. 1A, B). Upon leaving the ventricular wall, labeled profiles arranged in a curved path that paralleled the white matter, instead of entering the overlying cortex (Fig. 1C). Thus, labeling in this region appeared to represent DCX expression in the subventricular zone-rostral migratory stream, as characterized in other species (Nacher et al.,

Discussion

DCX or PSA-NCAM+ cells occur in the piriform and medial prefrontal cortices in small laboratory rodents (Seki and Arai, 1991, Fox et al., 2000, Nacher et al., 2001, Sairanen et al., 2007). These cells are reported or have been noticed in the neocortex as well in larger mammals, including guinea pigs (Xiong et al., 2008, Luzzati et al., 2008), rabbits (Luzzati et al., 2008), cats (Gómez-Climent et al., 2008), monkeys (Bernier et al., 2002, Kornack et al., 2005), and humans (Varea et al., 2007,

Acknowledgments

This study was supported in part by Southern Illinois University School of Medicine and Illinois Department of Public Health (X.X.Y.), Xiangya Medical School (Y.C., X.K.) and National Institute of Health (1R21NS056371 to P.R.P., X.X.Y. and NS054038 to A.W).

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    These authors contributed equally to this work.

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