Quantitative analysis of postnatal neurogenesis and neuron number in the macaque monkey dentate gyrus

Abstract

The dentate gyrus is one of only two regions of the mammalian brain where substantial neurogenesis occurs postnatally. However, detailed quantitative information about the postnatal structural maturation of the primate dentate gyrus is meager. We performed design-based, stereological studies of neuron number and size, and volume of the dentate gyrus layers in rhesus macaque monkeys (Macaca mulatta) of different postnatal ages. We found that about 40% of the total number of granule cells observed in mature 5-10-year-old macaque monkeys are added to the granule cell layer postnatally; 25% of these neurons are added within the first three postnatal months. Accordingly, cell proliferation and neurogenesis within the dentate gyrus peak within the first 3 months after birth and remain at an intermediate level between 3 months and at least 1 year of age. Although granule cell bodies undergo their largest increase in size during the first year of life, cell size and the volume of the three layers of the dentate gyrus (i.e. the molecular, granule cell and polymorphic layers) continue to increase beyond 1 year of age. Moreover, the different layers of the dentate gyrus exhibit distinct volumetric changes during postnatal development. Finally, we observe significant levels of cell proliferation, neurogenesis and cell death in the context of an overall stable number of granule cells in mature 5-10-year-old monkeys. These data identify an extended developmental period during which neurogenesis might be modulated to significantly impact the structure and function of the dentate gyrus in adulthood.

Figure 1

Coronal Nissl-stained sections at mid rostrocaudal level through the dentate gyrus of a newborn (A) and 7.7-year-old (B) macaque monkey (Macaca mulatta). ml: molecular layer; gcl: granule cell layer; pl: polymorphic layer. Scale bar: 500 μm in A, applies to panels A and B. C, Nissl-stained section illustrating the presence of a large population of immature neurons in the deep portion of the granule cell layer of a newborn monkey. D, Nissl-stained sections showing the same area in an adult monkey. Note the absence of the large population of immature neurons in the adult as compared to the newborn monkey. E, NeuN-immunostained section showing that the large population of cells identified as immature granule cells in Nissl preparations (C) are consistently, but faintly labeled with NeuN. F, NeuN-immunostained section showing the same area in an adult monkey. All neurons in the adult granule cell layer are heavily labeled with NeuN. Scale bar: 10 μm in C, applies to panels C, D, E and F.

Figure 2

Ki-67-immunohistochemistry. A, Number of Ki-67-positive cells in the molecular layer (white diamonds) and in the combined granule cell and polymorphic layers (black circles) of the macaque monkey dentate gyrus through early postnatal development and in adulthood. Note the intermediate level of cell proliferation between three months and one year of age. B, Photomicrograph of Ki-67-labeled cells in the granule cell and polymorphic layers of the dentate gyrus. Scale bar: 10 μm. Abbreviations, see Fig. 1.

Figure 3

Phenotype of BrdU-labeled cells in the macaque monkey dentate gyrus, four weeks following BrdU injection (150 mg/kg). A, Phenotype of BrdU-labeled cells in the combined granule cell and polymorphic layers. Note the significant rate of Brdu/NeuN-positive cells until one year of age. B, Confocal image of BrdU/NeuN-positive (yellow arrow) and BrdU/S100beta-positive (white arrowheads) cells at the border between the granule cell and polymorphic layers. Scale bar: 10 μm, applies to all panels. C, Phenotype of BrdU-labeled cells in the molecular layer. Note the absence of BrdU/NeuN-positive cells in the molecular layer. D, Confocal image of a BrdU/S100beta-positive cell (white arrowhead) in the molecular layer. The different panels in B and D represent different planes of section (1 μm apart) through the depth of the labeled cells. Scale bar: 10 μm, applies to all panels.

Figure 4

Representative coronal sections through the monkey dentate gyrus illustrating the location of Ki-67-positive cells (A–C), BrdU-positive cells four weeks after BrdU Injection (D–F) and TUNEL-positive cells (G–I), at different ages through postnatal life (A,D,G: newborns; B,E,H: 1-year-olds; C,F,I: 5–10-year-olds). Note the different distribution of Ki-67- and BrdU-labeled cells across the polymorphic and granule cell layers. Note also the location of TUNEL-positive cells across all layers of the dentate gyrus, including the granule cell layer. Scale bar: 500 μm in I, applies to all panels. Abbreviations, see Fig. 1.

Figure 5

TUNEL staining. A, Number of TUNEL-positive cells in the molecular layer (white diamonds) and in the combined granule cell and polymorphic layers (black circles) of the macaque monkey dentate gyrus through early postnatal development and in adulthood. B, Photomicrograph of a TUNEL-positive cell in the granule cell layer. Scale bar: 10 μm.

Figure 6

Total number of neurons in the granule cell layer (A) and volumes (B,C,D) of the different layers of the macaque monkey dentate gyrus through early postnatal development and in adulthood. Note the large increase in neuron number between birth and three months of age, and the gradual increase of about 3,100 neurons per day between three months and one year of age. B, Volume of the granule cell layer. C, Volume of the molecular layer. D, Volume of the polymorphic layer. Note the linear increase in volume of the granule cell and molecular layers between birth and one year of age. In contrast, the polymorphic layer exhibits a significant increase only past nine months and one year of age.

Figure 7

Bimodal distribution of neuronal soma size in the monkey granule cell layer through early postnatal development and in adulthood. Grey areas indicate the two modes: mode 1, corresponding to immature granule cells (soma < 150 μm³); mode 2, corresponding to the median size of mature granule cells (400–550μm³). A, newborns. B, 3-month-olds. C, 6-month-olds. D, 9-month-olds. E, 1-year-olds. F, 5–10-year-olds.