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 (). Upon leaving the ventricular wall, labeled profiles arranged in a curved path that paralleled the white matter, instead of entering the overlying cortex (). 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., 2001
; Gritti et al., 2002
; Xiong et al., 2008
). Immunoreactive perikarya and neuritic processes were also clearly present within the cortical mantle (). A cellular band composed of immunoreactive somata and processes was evident in layers II and upper III across all the frontal cortical regions assessed, and were most dense at the upper border of layer II. A small number of labeled cells also occurred in other cortical layers (). The ventral portions of the frontal lobe, including the ventral medial frontal area (VMF) (), dorsal medial prefrontal area (DMP) () and dorsal lateral prefrontal area (DLP) (), contained more labeled profiles relative to the dorsal (dorsolateral and dorsomedial) regions of this lobe occupied by the primary somatosensory and motor areas ().
Fig. 1 Doublecortin immunoreactive (DCX+) profiles in the frontal cortical areas in a 1.3 yr-old cat, as marked on the cerebral map in panel A. Panels B and C illustrate heavy labeling in the subventricular zone around the anterior horn of the lateral ventricle (more ...)
In the temporoparietal lobe sections, DCX expression was prominent in both the cortex and the subgranular zone (; Supplemental Fig. 1
). A clear ventrodorsal (high to low) gradient existed across the cortical hemisphere, especially regarding the abundance of labeled somata and processes in layers II/III. For instance, at the levels of LGNd, labeled profiles were most abundant in the entorhinal cortex but declined when moving towards the temporal, auditory II, auditory I, and finally the primary visual, areas (Supplemental Figs. 1C–G
). Similarly, at the level of the medial geniculate body, labeled cells and processes appeared very dense in the entorhinal and adjoining temporal areas, with a noticeable reduction of the labeling in the latter ().
Fig. 2 Doublecortin labeling in the entorhinal and adjoining temporal cortex of a young adult cat, focusing on clusters and chain-like formations. Panels A and B are low power views of immunolabeling and Nissl stain, respectively. Immunolabeled profiles in the (more ...)
In the occipital lobe sections, a ventrodorsal gradient in the amount of labeling occurred across the visual domain areas. Thus, labeled profiles appeared to decrease in number moving from area 20 (V4), to area 19 (V3), to area 18 (V2), and to area 17 (V1) (Supplemental Figs. 2A–H
DCX labeling was rare in the subventricular zone and adjacent white matter in proximity to the inferior and posterior horns of the lateral ventricle (; Supplemental Figs. 2A, B, D
), in sharp contrast with the pattern seen around the anterior horn ().
Decrease of DCX+ cells with age in cat cortex
DCX+ cells were detectable in most cortical areas in all of the older adult cats examined in the current study (3.5–5.2 yr-old). However, the abundance of labeling appeared to be reduced in general in these animals relative to young adults, while the overall laminar distribution pattern as well as the ventrodorsal gradient of labeling was largely maintained with age (, Supplemental Fig. 3
). Specifically, in the 5.2 yr-old cat (the oldest in this study), the cellular band in layers II/upper III remained visible in the ventromedial and ventrolateral frontal cortical areas (), the entorhinal and low temporal areas () and the ventral occipital regions (areas 20, 19) (not shown). This band was not evident in more dorsal cortical regions (both the lateral and medial aspects), such as S1, A2, A1, V2 and V1, as well as the motor cortex (not shown). However, as in the cortex of young animals, a small number of weakly stained cells remained in most cortical regions (). Consistent with a global decline of DCX expression in the brain, immunoreactivity in the frontal lobe subventricular zone and hippocampal subgranular zone was dramatically reduced in this adult cat ().
Fig. 3 Doublecortin immunoreactive (DCX+) cells in representative forebrain areas in a 5.2 yr-old cat, as indicated in panel A. Panels B–G illustrate occurrence of some DCX+ cells in the ventral frontal areas, mostly in layers II/III. These cells are (more ...)
Quantitative analysis was carried out to assess cell density in the cellular band over layers II and upper III, which exhibited apparent region and age-related alterations (). A montage of three adjoining 10× images was created for a given cortical region, which was imported as the background layer of a CorelDraw file. To define the vertical distance (depth) of the cellular band in a systematic manner, we used layer I as an internal reference. Thus, the depth of the band was set to be equivalent to that of the overlying layer I in the same area. Dots were created, group-copied and placed one by one on top of individual labeled somata. The background image layer was subsequently deleted, resulting in a map of labeled cells over the area of interest (’). Total area of the band (T), areas occupied by all dots (Tdt) and a single dot (dt) (i.e., unit area) were measured with Image-J. Cell density was thus calculated based on the formula Tdt/dt/T, and expressed as number of cells per mm2.
Fig. 4 Quantitative analyses of DCX+ cell density over the cellular band in layers II and upper III in selected cortical areas in young (mean age= 1.5 ± 0.3 yr-old) and older (4.5 ± 0.7 yrold) adult cats. Panels A–B’ illustrate (more ...)
An overall decline of DCX+ cells was found between the young and older adult groups of cats across all areas analyzed. As plotted in , the age-related difference was significant for the frontal areas (F = 32, DFn = 1, DFd = 15), temporoparietal areas (F =64, DFn=1, DFd= 20), and the occipital areas (F = 48, DFn= 1, DFd= 20) (with p<0.0001 in all lobes, two-way ANOVA)]. Also, there existed an overall difference among the analyzed cortical subregions of the same lobe in both age groups [F = 10 (DFn = 1, DFd= 15) for three frontal areas, F =11 (DFn = 1, DFd = 20), for four temporoparietal areas, and F = 14 (DFn = 1, DFd = 40) for four visual domain areas, p<0.005]. Of note, the following pair comparisons of means did not reach statistical significance in Bonferroni post-tests: S1 vs motor areas, A2 vs A1, V3 vs V2 or V1, and V2 vs V1 in the adult group, as well as V2 vs V1 in the young adult group ().
Overall morphology of DCX+ cells in cat cortex
DBX+ cortical cells in both the young and older adult cats exhibited a heterogeneous morphology largely in a lamina-dependent manner. Within the cellular band over layers II and upper III, DCX+ cells showed a great variability in size, shape, labeling intensity, and number/length/thickness of processes (Figs. ; ; Supplemental Figs. 1H–J; 2I, J
). Somal size of the cells ranged from considerably small (~5 μm) to fairly large (up to 20 μm). The small cells were mostly unipolar or bipolar, whereas larger ones often multipolar and some irregular (Figs. ; ; ). Larger cells generally had stronger immunoreactivity, thicker and longer neuritic processes relative to small cells. However, some large cells with well-formed processes exhibited otherwise apparently reduced immunoreactivity (Figs. ; ; Supplemental Figs. 1H, I; 2I
Fig. 5 Colocalizations of doublecortin (DCX) and polysialylated neural cell adhesion molecule (PSA-NCAM) (A–E), neuron-specific nuclear protein (NeuN) (F–J), γ-aminobutyric acid (GABA) (K–M) and glutamic acid decarboxylase (GAD)-67 (more ...)
Other labeled cells exhibited relatively uniform morphology characterized by medium size (10–15 μm), round/oval somal shape, weak reactivity and short processes. These cells were found in all cortical layers with a general low density (Figs. ; ; ). Moving from ventral to dorsal cortical regions at a given hemispheric level, these medium-sized cells became somewhat more prominent due to the reduced DCX+ cells in layers II/III (; Supplemental Figs. 1D–G; 2E–J; 3G, H
). Pilot densitometry failed to detect a statistically significant regional difference of these cells over layers III to VI (temporal lobe regions, young adult group). Therefore, no effort was made to quantify these cells in subsequent analysis.
Cluster and chain-like arrangements of DCX+ cells in cat cortex
DCX+ cells in layers II/III sometimes arranged as clusters that contained a few or several closely apposed somata (Figs. ; ; ). Cells within a given cluster often appeared to have comparable morphological characteristics, e.g., similar somal size and shape, staining intensity and neuritic complexity (Figs. ; ; ). However, in some cases closely apposed cells exhibited dramatic difference in reactivity and morphology (Figs. ; ).
A unique type of cell cluster consisted of small labeled cells and their processes seemingly arranged as migratory chains, which occurred largely in the entorhinal cortex and were most prominent in the young adult cats (Figs. ; ). A typical chain structure had an enlarged base located in layer II, and a long chain that became thinner as it extended into deeper layers (as far as layer V). The base was packed with many small cells, some of which appeared to migrate away in a radiation manner, either tangentially or obliquely (towards the white matter). The remainder of the cells appeared to migrate inwardly along the chain, with some voyaging away en route. The proximal portion of the chain (close to the base) was thicker and contained more densely packed cells relative to the distal portion. The latter consisted of a few fusiform somata that were separated with increasing length of labeled processes, especially towards the end of the chain (Figs. ; ).
In older adult cats, there was a dramatic reduction of cells associated with a given chain structure (; Supplemental Figs. 3D–F
). As a result, the base of the chain in layer II became much smaller or no longer impressive. Also, in most parts of the chain labeled somata and processes were aligned as a single array. As with those in young adult cats, cells appeared to migrate away from the chain in the proximal or distal portions. Cells that had left the chain appeared somewhat larger in size and exhibited weaker reactivity relative to their counterparts remaining on the chain (; Supplemental Fig. 3F
Colocalization of immature and mature neuronal markers in DCX+ cells in cat cortex
Double labeling was carried out in the cat cortex to assess DCX colocalization with other neuronal and glial markers similar to our recent characterization of these cells in guinea pigs (Xiong et al., 2008
). DCX+ cells in layers II–III exhibited virtually a complete colocalization with PSA-NCAM () and TuJ1 (not shown).
NeuN and GABA were differentially expressed among DCX+ cells (). Thus, small unipolar and bipolar DCX+ cells, including those arranged in clusters and chain-like formations, did not exhibit NeuN or GABA reactivity. In contrast, medium to large DCX+ cells in bipolar and multipolar shapes and with well-developed neuritic processes consistently co-expressed NeuN and GABA. Of note, all medium-sized DCX+ cells, including those located deep to the cellular band in layers II/III, displayed clear colocalization with NeuN and GABA (). A clear transition in relative levels of DCX and NeuN could be found among a group of cells within a local area. Thus, DCX levels appeared to increase as the cells became larger and had more and thicker neuritic processes. However, DCX levels attenuated as the cells became even more mature-looking, in parallel with emergence and elevation of NeuN ().
Medium and large-sized cells with attenuated DCX reactivity were specifically found to colocalize partially with several other terminal markers of interneurons. Thus, DCX+/GAD67+ cells occurred in layers II/III as well as in deeper layers (), as were DCX+/CB+ cells (). DCX+/PV+ cells were found in layers II/III but not in deep cortical layers, with DCX reactivity in these double-labeled cells being faint or very weak (). No DCX and CR double-labeled cells were found in the present study (). Colocalization of NADPH-d or nNOS appeared to be considerably common in medium-sized cells with weak DCX reactivity localized to the cellular band over layers II and upper III as well as the remainder of the cortex (). In fact, DCX+/NADPH-d+ cells were also found in layer I (not shown). All of these double-labeled cells were type II NADPH-d neurons (Yan et al., 1996b
; Estrada and DeFelipe, 1998
; Garbossa et al., 2005
). Of note, DCX+/NADPH-d+ cells appeared somewhat smaller than neighboring type II neurons that lacked DCX but exhibited stronger NADPH-d reaction ().
Fig. 6 Colocalizations of calcium-binding proteins in DCX+ cells in the temporal neocortex of an adult cat (3.5 yr-old). Panels A–C show a partial colocalization (white arrows) of calbindin (CB) in medium (A, B) to large (C, with boxed areas enlarged (more ...)
Fig. 7 Colocalization of DCX with β-nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) or neuronal nitric oxide synthase (nNOS) in adult cat (3.5 yr-old) cerebral cortex. Panels A–F show images from batch-processed sections with (more ...)
Some medium-sized DCX+ cells in layers III–V also colocalized with somatostatin (SOM) (Varea et al., 2007
). No DCX+ cells were found to express neurogranin, a marker of pyramidal neurons (Xiong et al., 2008
). Labelings for GFAP (astrocytes), OX42 (microglia) and oligodendrocyte-specific protein, were not detected in any DCX+ cell (data not shown).
Presence of similar DCX+ cells in adult monkey and human cortex
Morphologically heterogeneous DCX+ cells were observed in layers II and upper III in most cortical areas of all the monkeys examined (). In the frontal lobe, a cellular band at this location appeared in the ventral and medial cortical regions especially the prefrontal areas (), whereas individual labeled somata were detectable in the remaining regions (not shown). Most labeled cells were bipolar but some multipolar. Larger and weakly stained cells were visible in deeper cortical layers (). In the temporoparietal cortex, numerous cells occurred in the entorhinal area, and they arranged as large clusters seemingly associated with the island formations of layer II (). Labeled profiles formed virtually a continuous band in layers II/upper III throughout the temporal gyri (), which extended dorsally into the insular and adjoining parietal cortex, with an apparently reduced cell density (). A few labeled cells occurred further dorsally in the primary somatosensory and motor areas (). In the occipital lobe, a few cells were encountered over layer II in areas 17, whereas more cells at this lamina occurred in the associative visual areas peripheral to the striate cortex (not shown). Moderate DCX expression was present in the dentate subgranular zone of these monkeys ().
Fig. 8 Doublecortin immunoreactive (DCX+) cells in representative adult monkey (Macaca mulatta) forebrain areas, as marked on the upper-left hemispheric maps (A). Panels B–D illustrate DCX+ cells in the prefrontal areas. Distinctly and weakly stained (more ...)
DCX+ cells were detectable in the cortex of all human specimens examined in this study (). Labeled cells at the upper border of layer II varied considerably in somal size and shape, staining intensity and neuritic appearance, with small bipolar and a few multipolar cells exhibited heavy reactivity (). Medium-sized cells with weak to moderate reactivity were found in layers II–VI (mostly in II–IV) (). Because the density of labeled cells varied greatly between cases, no quantitative analysis was included in the current study.
Fig. 9 Doublecortin immunoreactive (DCX+) cells in representative adult human temporal (A–F) and frontal (G–M) cortices prepared from surgically removed biopsies. Distinctly stained DCX+ cells exist in layer II, most of which are small bipolar (more ...)