The Journal of Neuroscience, February 1, 2003 • 23(3):937–942 • 937 Nonrenewal of Neurons in the Cerebral Neocortex of Adult Macaque Monkeys Daisuke Koketsu,1 Akichika Mikami,2 Yusei Miyamoto,1 and Tatsuhiro Hisatsune1 1Department of Integrated Biosciences, University of Tokyo, Kashiwa 277-8562, Japan, and 2Primate Research Institute, Kyoto University, Inuyama 484-8506, Japan The concept that, after developmental periods, neocortical neurons become numerically stable and are normally nonrenewable has been challengedbyareportofcontinuousneurogenesisintheassociationareasofthecerebralcortexintheadultMacaquemonkey.Therefore, we have reexamined this issue in two different Macaque species using the thymidine analog bromodeoxyuridine (BrdU) as an indicator of DNA replication during cell division. We found several BrdUϩ/NeuNϩ (neuronal nuclei) double-labeled cells, but cortical neurons, distinguished readily by their size and cytological and immunohistochemical properties, were not BrdU positive. We examined in detail the frontal cortex, where it is claimed that the largest daily addition of neurons has been made, but did not see migratory streams or any sign of addition of new neurons. Thus, we concluded that, in the normal condition, cortical neurons of adult primates, similar to other mammalian species, are neither supplemented nor renewable. Key words: adult neurogenesis; cerebral cortex; primates; DNA synthesis; glial markers; 3D immunohistochemistry Most neurons of the mammalian brain are generated from neu- Gould and colleagues assert, on the basis of labeling with the roepithelial cells near cerebral ventricles during well defined de- thymidine analog bromodeoxyuridine (BrdU), that streams of velopmental stages and then act as substrates of neural circuitry new neurons are generated continuously and added to the asso- throughout life (Rakic, 1985). However, recent findings of adult ciation neocortex of adult Macaque monkeys (Gould et al., neurogenesis in selected structures of the mammalian brain 1999b), where many form connections and survive for weeks present the possibility that this concept does not apply for all before dying (Gould et al., 2001). The daily addition of thousands brain regions, in particular, the neural circuits of the olfactory of new neurons to the principal sulcus alone (Gould et al., 1999b) bulb and dentate gyrus of the hippocampus, which may be in the absence of net growth indicated high turnover. To verify changing their basic circuitry with the arrival of newly born neu- this claim, Kornack and Rakic (2001a) have performed experi- rons (Rochefort et al., 2002; van Praag et al., 2002). In addition, ments using the BrdU method in adult Macaque monkeys. They the application of new staining methods in the adult Macaque found BrdU-labeled non-neuronal cells, but they did not detect monkeys uncovered some new neurons in the olfactory bulb newly born neurons in the neocortex, nor did they find the (Kornack and Rakic, 2001b) and dentate gyrus of the hippocam- stream of migrating neurons from the proliferative subventricu- pus (Gould et al., 1999a; Kornack and Rakic, 1999). lar zone to the principal sulcus of the prefrontal cortex (Kornack In terms of the cerebral neocortex, studies in various mam- and Rakic, 2001a). This discrepancy in basic findings required a malian species that range from mice and rats to ferrets and cats reexamination in other laboratories (Nowakowski and Hayes, have indicated that neurogenesis in this structure stops after well 2000). defined developmental periods (Hicks and D’Amato, 1968; Cavi- Therefore, we addressed the question of whether new neurons ness and Sidman, 1973; Luskin and Shatz, 1985; Jackson et al., are generated and continuously added in the neocortex of juve- 1989; Takahashi et al., 1996). Furthermore, no sign of neuronal nile and young adult Macaque monkeys using BrdU methods for renewal has been found in the cortex of adult rodents (Magavi et labeling DNA synthesis in the dividing brain cells (Nowakowski al., 2000). An extensive analysis of Macaque monkeys exposed to [ 3H]thymidine indicated that the neocortical neurons in pri- et al., 1989) that were used by previous investigators. We exam- mates are generated prenatally (Rakic, 1974) and not normally ined in detail the frontal cortex, where it has been previously renewed during adulthood (Rakic, 1985, 2002a). It was suggested claimed that the largest daily addition of neurons is made (Gould that a stable population of neurons in the cortex might be an et al., 1999b). We selected juvenile and young adult monkeys important mechanism for continuity of learning and preserving because they presumably have more neuronal plasticity and to memory over a lifetime (Rakic, 1985). Contrary to this theory, avoid the possibility of BrdU labeling during unscheduled DNA synthesis in the process of cell death (apoptosis) in aged animals (Yang et al., 2001). We also avoided high doses of BrdU to avoid Received Sept. 13, 2002; revised Nov. 4, 2002; accepted Nov. 20, 2002. This study is supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports its possible mutagenic and stimulation effect on DNA synthesis andCultureofJapan.WethankDr.JoseAntonioCampos-OrtegaandDr.YukioHirataforinformativediscussionand (Nowakowski and Hayes, 2001; Rakic, 2002b). To identify the technical advice. We also thank Dr. Shinichi Kohsaka for kindly providing an anti-Iba-1 antibody. phenotypes of BrdU-labeled cells in the neocortex, an immuno- Correspondence should be addressed to Tatsuhiro Hisatsune, Department of Integrated Biosciences, Graduate histochemical analysis was performed with neuronal markers School of Frontier Sciences, The University of Tokyo, Bioscience Building 402, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan. E-mail: [email protected]. NeuN, a transcriptional factor that is expressed in the nucleus Copyright © 2003 Society for Neuroscience 0270-6474/03/230937-06$15.00/0 and cytoplasm of neurons (Mullen et al., 1992), and doublecortin 938 • J. Neurosci., February 1, 2003 • 23(3):937–942 Kotetsu et al. • Stability of Cortical Neuron in Adult Primates tions were preserved in a cryoprotectant solution (30% ethylene glycol, 30% glycerol in 0.05 M phosphate buffer) at Ϫ20°C until they were processed. Immunohistochemistry. Sections were washed twice with Tris-buffered saline (TBS) for 10 min. Sections were then treated in 10 M citric acid buffer at 90°C for 5 min. The heated sections were left at room temper- ature for 30 min and washed with TBS for 10 min. The sections were incubated in 1 M HCl at 37°C for 30 min and neutralized by rinsing with 0.1 M borate buffer and washed twice with TBS. The sections were then Figure1. BrdUinjectionschedule.ThedayofBrdUinjectionisrepresentedbythefilledcircle, blocked with a blocking solution (5% normal donkey serum and 0.3% and the day of perfusion is represented by the arrowhead. M. fascicularis I received once-daily Triton X-100 in TBS) for 30 min at room temperature with gentle shak- BrdU injections for 3 consecutive days and one more injection after 7 d and then was perfused ing. The blocked sections were reacted with primary antibodies at 4°C. 1 d after the final injection. M. fascicularis II received five once-daily injections every other day The concentration of Triton X-100 in the blocking solution dissolving and then was perfused 14 d after the final injection. M. fuscata I and M. fuscata II received primary antibodies with 1 or 3 d reaction time was 0.3 or 0.1%, respec- once-dailyinjectionsfor5consecutivedays.Then,M.fuscataIandM.fuscataIIwereperfused25 tively. Monoclonal rat anti-BrdU antibody (Harlan, Leicestershine, UK) or26dafterthefinalinjection,respectively.Thenumberofdaysrepresentstheperiodbetween with a 1:200 dilution and monoclonal mouse anti-BrdU (Becton Dick- the first injection and perfusion. inson, Franklin Lake, NJ) at 1:33 were used. Some antibodies to identify cell types were also used. As antibodies to the neuronal marker, mono- clonal mouse anti-NeuN antibody (1:1000; Chemicon, Temecula, CA) (DCX) (Gleeson et al., 1999; Nacher et al., 2001) and also with and polyclonal goat anti-DCX antibody (1:100; Santa Cruz Biotechnol- markers of other non-neuronal cell types. GFAP and S-100␤ (as- ogy, Santa Cruz, CA) were used. The reaction time of NeuN was1dand troglial cell), O4 (oligodendrocyte), and Iba-1 (microglial cell) that of DCX was 3 d. As antibodies to the astroglial marker, polyclonal (Ito et al., 1998) were used. rabbit anti-GFAP antibody (1:10,000; Dako, Carpinteria, CA) and poly- clonal rabbit anti-S-100␤ antibody (1:5000; Swant, Bellinzona, Switzer- Materials and Methods land) were used. As the antibody to oligodendrocyte marker, monoclonal Animals. Two young adult female Macaca fascicularis (Cynomolgus mouse IgM anti-O4 antibody (1:10; Chemicon) was used. Then, as the monkey; 5 years of age) and two juvenile female M. fuscata (Japanese antibody to microglial marker, polyclonal rabbit IgG anti-Iba-1 antibody monkey; 2 years of age) were used. Monkeys were housed at the Primate (1:250; kind gift from S. Kohsaka, National Institute of Neuroscience, Research Institute of Kyoto University and were maintained in individ- Tokyo, Japan) was used. The reaction time of GFAP, O4, and Iba-1 was ␤ ual cages with up to 16 cages facing each other in a single room. Thus, the 1 d and that of S-100 and O4 was 3 d. After immunoreaction with these monkeys could see and interact with other monkeys. In addition, Japa- primary antibodies, sections were washed with TBS. The sections were nese monkeys were provided with a piece of wood that was hung in the then reacted with fluorochromo-conjugated secondary antibodies. As cage as a playing tool and a food pickup toy that was placed in front of the secondary antibodies, Alexa 488 (1:1000; Molecular Probes, Eugene, OR) cage every day for several hours. The size of the toy was 60 ϫ 60 cm with and rhodamine Red-X (1:200; Jackson ImmunoResearch, West Grove, 480 holes, half of which were covered with a piece of plate so that the PA) were used.