Doublecortin-Expressing Neurons in Chinese Tree Shrew Forebrain Exhibit Mixed Rodent and Primate-Like Topographic Characteristics

Abstract

Doublecortin (DCX) is transiently expressed in new-born neurons in the subventricular zone (SVZ) and subgranular zone (SGZ) related to adult neurogenesis in the olfactory bulb (OB) and hippocampal formation. DCX immunoreactive (DCX+) immature neurons also occur in the cerebral cortex primarily over layer II and the amygdala around the paralaminar nucleus (PLN) in various mammals, with interspecies differences pointing to phylogenic variation. The tree shrews (Tupaia belangeri) are phylogenetically closer to primates than to rodents. Little is known about DCX+ neurons in the brain of this species. In the present study, we characterized DCX immunoreactivity (IR) in the forebrain of Chinese tree shrews aged from 2 months- to 6 years-old (n = 18). DCX+ cells were present in the OB, SVZ, SGZ, the piriform cortex over layer II, and the amygdala around the PLN. The numerical densities of DCX+ neurons were reduced in all above neuroanatomical regions with age, particularly dramatic in the DG in the 5-6 years-old animals. Thus, DCX+ neurons are present in the two established neurogenic sites (SVZ and SGZ) in the Chinese tree shrew as seen in other mammals. DCX+ cortical neurons in this animal exhibit a topographic pattern comparable to that in mice and rats, while these immature neurons are also present in the amygdala, concentrating around the PLN as seen in primates and some nonprimate mammals.

Keywords: adult neurogenesis; cerebrum; immature neurons; mammalian evolution; neuroplasticity.

Figure 1

Doublecortin immunoreactive (DCX+) profiles in the rostral part of the forebrain in a 2 months-old Chinese tree shrew. Panels (A–C) are low magnification images showing the labeling in the olfactory bulb (OB) at the levels of the anterior olfactory nucleus (AON) (A), the beginning of piriform cortex (Pir) (B), and septum (Sept) (C), respectively. Framed areas are enlarged and illustrated as (D–I). In the OB, DCX+ cells are densely packed at the subventricular zone (SVZ) surrounding the olfactory ventricle (OV). Moderately stained cells are present over the subependymal zone (SEZ) and granule cell layer (GCL) (D). DCX+ granule cells are mostly bipolar or fusiform with their somata and processes oriented centroperipherally (E). Panels (F,G) are higher magnification images illustrating DCX+ cellular chains forming the rostral migratory stream (RMS) from the SVZ in the frontal lobe. Panel (H) shows part of the frontal cortex (FC) and Pir surrounding rhinal fissure (RF), with no labeled cells in the former, whereas a few labeled cells in the latter (insert). Panel (I) shows the lack of DCX+ cells in the parental neocortex (PC). Additional abbreviations: CC, corpus collosum; St, striatum; WM, white matter; MCL, mitral cell layer; I, II…VI: cortical layers. Scale bars are as indicated in individual image panels.

Figure 2

DCX+ cellular profiles in the middle part of the forebrain in a 2 months-old Chinese tree shrew. Panels (A,B) are low magnification views of immunolabeled hemispherical sections at the levels passing the rostral end of the hippocampus (Hipp) and the lateral geniculate nucleus (LGN), respectively, with framed areas enlarged as panels (C–H). No labeled cells are seen in the parietal neocortex (PC) and the majority of the temporal neocortex (TC) (C,D), while a few cells could be detected in the temporal cortex surrounding the rhinal fissure (RF) (D, pointed by arrows). In contrast, a large number of labeled cells occurs in the piriform cortex (Pir) over layer II (D–F). Abundant labeling is present in the amygdala (Amyg) near its border to the white matter of the adjoining piriform and temporal cortices (F). DCX+ neurons and processes are distinctly present at the subventricular zone (SVZ) next to the lateral ventricle (LV) and in the subgranular zone of the dentate gyrus (DG) (G,H). Additional abbreviations: Th, thalamus; LF, lateral fissure; CA1, Ammons’ horn CA1 sector; SC, superior colliculus; RN, red nucleus; SN, substantia nigra; WM, white matter; PLN, paralaminar nucleus; ML, molecular layer; GCL, granule cell layer; Hi, hilus. I, II…VI: cortical layers. Scale bars are as indicated.

Figure 3

DCX+ cellular profiles in the caudal part of the forebrain in a 2 months-old Chinese tree shrew. Panels (A,B) are low magnification views of immunolabeled hemispherical sections at two caudal levels of the hippocampus, with framed areas enlarged as panels (C–H). DCX+ neurons in the dentate gyrus are separately located at the most dorsal and ventral parts of the hippocampal formation (C,D). Again, DCX+ neuronal somata and processes are not found in the temporal and parietal cortical areas (E,F), but are present in the piriform cortex over layer II (G). DCX+ cells are also found at the subventricular zone in the temporal and occipital lobes (H). Abbreviations are as defined in Figures 1, 2, and the following: cp, cerebral peduncle; hTh, hypothalamus; M, mammillary body; OC, occipital neocortex; MGN, Medial geniculate nucleus; RN, red nucleus; Aq, aqueduct; PAG, periaqueductal gray. Scale bars are as indicated.

Figure 4

Representative images illustrating DCX+ cellular profiles in the forebrain in a 2 years-old Chinese tree shrew. Panel (A) shows the labeling in the olfactory bulb (OB) and rostral migratory stream (RMS) at four coronal levels, noting the open olfactory ventricle (OV) inside the bulb. Panel (B) shows the labeled cells densely packed at the subventricular zone (SVZ), with migrating granule cells from the subependymal zone (SEZ) to the granule cell layer (GCL). Panel (C) shows labeled cells in the hippocampal dentate gyrus (DG), which are developing granule cells primarily resided along the subgranular zone (SGZ) (D). Panels (E,F) show DCX+ immature neurons localized selectively in the piriform cortex (Pir) over layer II, but not in the adjoining temporal neocortex. Panels (G–I) show high power views of DCX+ neurons in the piriform cortex and amygdala (Amyg), which are remarkably heterogeneous in somal size, shape, and labeling intensity. Other abbreviations are as defined in Figures 1–3. Scale bars are as indicated.

Figure 5

Representative images illustrating DCX+ cellular profiles in the forebrain in a 6 years-old Chinese tree shrew. Overall, the amount of labeling in the forebrain structures is apparently reduced relative to that seen in Figure 4. Panels (A–D) are low and high magnification images illustrating the labeling in the olfactory bulb (OB) in the granule cell layer (GCL), and along the rostral migratory stream (RMS). Panels (E–G) are low and high magnification images at the level of the septum. There are no labeled cells in the temporal and parietal neocortex (E,F). However, some lightly stained neurons are seen in the piriform cortex in layers II (E,G), while some distinctly labeled neurons remain in the amygdala (E,H). Panel (I) is the image of the immunolabeled section passing the temporal hippocampus, with labeled DCX+ cells seen in the piriform cortex (J) and a few clusters of labeled granule cells remained in the dentate gyrus (K,L). Abbreviations are as defined in Figures 1–3. Scale bars are as indicated.

Figure 6

Quantification of age-related decline of DCX+ immature neurons in the forebrain of Chinese tree shrews. Panels (A–C) illustrate the methodology for cell count in randomly determined zones in the piriform cortex (Pir) (A), amygdala (Amyg) (A), dentate gyrus (DG) (B), and the olfactory bulb (C). Counting zones (200 × 200 μm²) intersecting with layer II in the Pir are randomly generated based on the distance scales from the piriform sulcus to the ventral border of the cerebrum (marked with blue lines and arrows in A). Counting zones (100 × 100 μm²) in the Amyg are randomly generated in a rectangular area (boxed with blue lines) covering the entire amygdalar complex (pink circle). Only the zones (marked in green) within this tear-drop-shaped nucleus are considered as effective samples, whereas the zones (marked in red) outside its anatomical border are considered as ineffective sampled sites. Similarly, the counting zones (100 × 100 μm²) intersecting with the subgranular zone (SGZ) in the DG are randomly generated based on the distance scales from dorsal and ventral edges of the granule cell layer (GCL) (B). The effective sampling zones inside the bulb are across the granule cell layer (GCL) and the subependymal zone (SEZ) (pink circle), which are generated randomly within a rectangular area (marked with blue lines) covering the entire bulb. Panel (D) plots the means of DCX+ cell densities in the four regions from individual animals arranged in the three age groups (n = 4 in the 2–3 mo-old group, n = 5 in 1–2 yr-old group, and n = 5 in the 5–6 yr-old group). The normalized means relative to the youngest group are also provided in the graphs. Statistical results and significant intergroup differences (*) based on the one-way ANOVA test with Bonferroni’s pair-wise multiple comparisons are as indicated in individual graphs.

Figure 7

Double immunofluorescent characterization of DCX colocalization with the neuron-specific nuclear antigen (NeuN) and Ki67 in layer II of the piriform cortex (Pir) and paralaminar nucleus (PLN) of the amygdala (Amyg), with bisbenzimide (Bis) used to mark the cell nuclei (assessed in 2 years-old adult tree shrews). In both areas, there exists a partial colocalization of NeuN among the DCX+ cells (A–H, examples are pointed by arrows), whereas no Ki67 labeling is seen in the DCX+ cells (I–P). Image locations, fluorescent channels, and scale bars are as indicated in the panels.

Figure 8

Topographic distribution of DCX+ cellular profiles in sagittal cerebral sections from a 6-month-old animal, with hematoxylin counterstain. The approximate medial to lateral planes of the sections in reference to the top view of the brain are marked with a red line in the image panels. Framed areas in the low magnification images are enlarged as indicated. The area of the amygdalar complex (Amyg) is marked with broken purple lines (C–F). Panels (A,B) show the immunolabeling in the subventricular zone (SVZ) forming the rostral migratory stream (RMS) extending into the olfactory bulb (OB). Panels (C,D) show the distribution of DCX+ neurons in the hippocampal dentate gyrus (DG), Amyg, and piriform cortex (Pir), with the framed areas enlarged as (C1,C2), and (D1–D3) as indicated. Panels (E,F) are images at more lateral planes, in which the DCX+ cells in the Amyg are distinctly present in the area of the paralaminar nucleus (PLN) (E1,F1). Additional abbreviations: FC, frontal neocortex; PC, parietal neocortex; OC, occipital neocortex; Th, thalamus; hTh, hypothalamus; St, striatum; Sub, subiculum; GCL, granule cell layer; ML, molecular layer; Hi, hilus; WM, white matter. Scale bars are as indicated.

Figure 9

Topographic distribution of DCX+ cellular profiles in hematoxylin-counterstained horizontal cerebral sections from a 4-year-old animal. The approximate levels of the sections relative to the lateral view of the brain are marked with a red line in the image panels. Framed areas in the low magnification panels are enlarged as indicated. The area of the amygdalar complex (Amyg) is marked with broken purple lines (C,D). In the most dorsal level section, DCX labeling is seen at the subventricular zone lining the lateral ventricle surrounded by the stratum (St) and cingulate cortex (CgC) (A,A1,A2). There are no DCX+ cells in the frontal neocortex (A3) and subiculum (A4), with no specific labeling likely related to the meninge (A3,A4). In the section at the level of the olfactory bulb (B), DCX+ cells are found across the granule cell layer of the bulb (B1), the subgranular zone of the dentate gyrus (B2). Note that in the piriform cortex (Pir), no DCX+ cells are found in the rostral segment (B3), while a few cells are present in the caudal segment near the rhinal fissure (RF) (B4). At the basal levels of the cerebrum, a large number of DCX+ cells occur in the PLN of the amygdala (C1), while labeled cells are also abundantly present over layer II in the Pir (D1). Additional abbreviations are as defined in Figure 8. Scale bars are as indicated.