The generation, migration, and differentiation of olfactory neurons in the adult primate brain

Abstract

In adult rodents, neural progenitor cells in the subependymal (SZ) zone of the lateral cerebral ventricle generate neuroblasts that migrate in chains via the rostral migratory stream (RMS) into the olfactory bulb (OB), where they differentiate into interneurons. However, the existence of this neurogenic migratory system in other mammals has remained unknown. Here, we report the presence of a homologue of the rodent SZ/RMS in the adult macaque monkey, a nonhuman Old World primate with a relatively smaller OB. Our results-obtained by using combined immunohistochemical detection of a marker for DNA replication (5-bromodeoxyuridine) and several cell type-specific markers-indicate that dividing cells in the adult monkey SZ generate neuroblasts that undergo restricted chain migration over an extended distance of more than 2 cm to the OB and differentiate into granule interneurons. These findings in a nonhuman primate extend and support the use of the SZ/RMS as a model system for studying neural regenerative mechanisms in the human brain.

Figure 1

Dividing and newly generated cells in the SZ and OB of adult macaque monkeys as detected by immunoperoxidase staining for BrdU or PCNA. Examples of clusters of BrdU-labeled cells 2 h after the last of five daily injections (a and b) and of PCNA-positive cells (c and d) in coronal sections of the anterior SZ along the striatal wall of the lateral ventricle (LV). (a) Although occasional immunopositive cells initially appeared to be part of the ependymal layer (E), further examination revealed them to be immediately subjacent to it and, thus, to belong to the SZ; no evidence for cell proliferation in the ependymal layer was observed. (e and f) PCNA-positive cells (arrows) among a stream of cells in the SZ closely associated with the basal aspect of the anterolateral ventricle. C, caudate nucleus; S, septal nuclei. Upper left area is dorsal. (f) The boxed field in e at higher magnification, showing that PCNA-positive cells are in the SZ but not the ependyma. (g) A sagittal section of the OB shows “strings” of elongated, PCNA-positive nuclei (arrows) in the white matter (WM) as it enters the core of the OB. GL, granule cell layer. (h and i) BrdU-labeled mitotic figures in the OB 2 h after five daily injections. (h) An early anaphase cell in the glomerular layer. (i) A late anaphase/early telophase cell in the external plexiform layer. [Bars = 20 μm (a–d and f), 100 μm (e), 50 μm (g), and 10 μm (h and i).]

Figure 2

Newly generated cells in the anterior SZ of the adult macaque display a neuroblast phenotype as revealed by triple-label immunofluorescence and confocal microscopy. (a–c) A sagittal section through the striatal wall of the LV provides an oblique view of the anterior SZ, revealing an extensive network of TuJ1-positive cells (red) that are distributed singly or in chain-like aggregates. Imaging in the same x, y, and z registration reveals that many of these cells are colabeled with BrdU (b and c, green) and are closely associated with GFAP-positive fibers (c, blue). The TuJ1-positive chains do not extend into the adjacent caudate nucleus (CN) or overlying cortical white matter. (d–f) The same field partially demarcated by the box in c at higher magnification shows chains and individual BrdU-labeled neuroblasts (arrows) and their proximity to the GFAP-immunopositive ependymal lining (E in f). A BrdU-labeled cell (arrowhead) that is immunonegative for both TuJ1 and GFAP may be a “type C” precursor. (g–i) A 0.6-μm-thick optical section of the same BrdU-labeled neuroblast indicated by the crossed arrow in d–f, confirming that the BrdU signal (h and i, green) is confined to the nucleus of the TuJ1-labled cell rather than to an adjacent TuJ1-negative cell. (j–l) An example of a TuJ1-postitive cell with a BrdU-labeled nucleus (arrowhead), exhibiting a bipolar morphology similar to that of a migrating neuron. A growth cone-like swelling (arrow) appears at the end of the putative thick “leading” process, and a thinner process “trails” behind. a–i show labeling 75 days after BrdU injections; j–l, 97 days after BrdU injections. (Bars = 50 μm.)

Figure 3

The rostral migratory stream in the adult macaque monkey olfactory peduncle (OP). (a) A sagittal plane reveals that BrdU-labeled nuclei (arrows, green) are aligned in a strict linear pattern within the core of the peduncle. GFAP immunoreactivity (blue) in the OP is especially pronounced along this pathway. These BrdU-labeled nuclei are immunonegative for the mature neuronal marker, NeuN (red), unlike the neurons seen in the orbitofrontal cortex (OFC) and the few scattered neurons at the periphery of the peduncle that belong to the anterior olfactory nucleus. (b–d) Images in same x, y, and z registration show chains of neuroblasts in the rostral migratory stream. Within the peduncle, chains of intensely stained TuJ1-positive cells (red) are restricted to the core and are aligned parallel to the longitudinal axis. (c) Some cells within these chains have elongated BrdU-positive nuclei (arrows, green) oriented in the presumed axis of migration. (d) These chains are immediately surrounded by a sheath of GFAP-positive fibers (blue) but are GFAP-negative. (e–g) The TuJ1-positive chains in the peduncle (e, red) also coexpress PSA-NCAM (f, green) as indicated by yellow fluorescence when the two signals are merged (g). [Bars = 100 μm (a) and 25 μm (b–g).]

Figure 4

Newly generated cells in the adult macaque monkey OB 97 days after the last of five BrdU injections. (a–d) Granule neurons in the OB express NeuN (red), and astrocytes express GFAP (blue). (a and b) A cell in the granule cell layer that is labeled with BrdU (arrow, yellow green in b) also expresses NeuN (arrow, a), indicating a newly generated granule neuron. (c and d) An example of two BrdU-labeled nuclei (d, arrows, green) that did not emit a red fluorescence signal (c, arrows), demonstrating that the BrdU signal did not bleed into the red channel; these might be progenitors or newly generated nonneuronal cells. (Bars = 20 μm.)