Adult neurogenesis in mammals: an identity crisis.

The study of neurogenesis in the adult brain is among the most exciting and fastest moving areas of neuroscience today. In contrast to the high rate of neurogenesis in some vertebrates (Nottebohm, 2002), unambiguous evidence for new neurons in normal adult mammals ranging from rodents to primates has been confined to the dentate gyrus and olfactory bulb (Lois and AlvarezBuylla, 1964; Kaplan and Hinds, 1977; Kempermann et al., 1996; Kornack and Rakic, 1999, 2001a,b). These may serve as important model systems from which we can learn to introduce new neurons into more resistant brain structures (Rakic, 1998). However, the compelling desire for curing neurological disorders has fostered an uncommon willingness to accept unsound evidence for adult neurogenesis under normal and experimental conditions. Therefore, it is important to review the methods for identification of new neurons in the adult brain. The realization that nerve cells in the adult are not renewed initially came from the observed paucity of mitotic divisions and a lack of transient forms from simple to more complex neuronal morphologies (Ramón y Cajal, 1928). The introduction of the H-thymidine (H-dT) autoradiographic method for detecting cell division revealed that, unlike most somatic cells that are continuously renewed or can be regenerated, neurons behave as a nonrenewable epithelium (Leblond, 1964). H-dT is incorporated into nuclear DNA during the S phase of the cell cycle, and the amount of incorporation in a given cell is directly proportional to the number of silver grains overlaying it; a high level of silver grains in autoradiograms, after a single intraventricular injection, can be used as a sign of the “birthday” of a neuron (Fig. 1a,b). In macaque monkey after intraventricular injection, H-dT is present in the bloodstream for 10 min, and thus provides a highly precise tool for dating the time of last cell division (Nowakowski and Rakic, 1974). Neurons are considered new only if the number of overlaying silver grains is at least 50% of that recorded in maximally labeled nuclei in the same specimen (Rakic 1973, 1977). Such a stringent criterion is needed to avoid misinterpreting light labeling caused by background or other artifacts as a sign of cell division (Nowakowski and Hines, 2000, 2002; Rakic, 2002). Indeed, the presence of both heavily and lightly labeled cells has been used as a criterion for confirming that the detected DNA synthesis is actually caused by cell proliferation (Angevine, 1965). Over the past few decades H-dT autoradiography has been used as a method of choice for determination of the time of neuron origin in mammals. The most comprehensive data are available for rodents (rat) and primates (macaque monkey). All mayor classes of neurons in the structures that range from the spinal cord to the cerebral cortex have been examined, and their survey is beyond the scope of this mini-review. For the purpose of the present article, it is sufficient to state that with the exception of interneurons in the olfactory bulb and dentate gyrus, each CNS structure in both of these mammals was found to acquire its neurons during a specific developmental time window. Importantly, the duration of this time window is not related to the final quantity of neurons in a given structure [e.g., 1.2 million retinal ganglion cells in macaque monkey are generated during 40 d (E30–E70), whereas 1.5 million geniculate neurons are produced in 8 d (E36–E43) (Rakic, 1977; LaVail et al., 1991)]. Altogether, the results of these studies indicate that a species-specific size of a given structure is determined early in the proliferative zone by genes controlling cell production (i.e., onset, cessation, length of cell cycle, symmetric–asymmetric mode of cell division, and the rate of programmed cell death) as well as by regulation of the allocation of postmitotic cells by the gradients of attractive and repulsive molecules. These findings in mammals stand in contrast to the high rate of neurogenesis in many adult vertebrates (Nottebohm, 2002). Thus, overcoming the resistance of the brain to the acquisition of functionally competent new neurons will require an understanding of why neurogenesis ceases at the end of specific developmental time windows and why there are species-specific and regional variations in this phenomenon (Rakic, 2002). More recently, evidence for neurogenesis is being obtained by the use of the thymidine analog bromodeoxyuridine (BrdU), which also incorporates into DNA during S phase (Nowakowski et al., 1989). The advantage of this method is that its immunohistochemical detection is more easily combined with that of various cell class-specific markers to determine cell phenotype in small neurons such as granule cells that are difficult to distinguish from astrocytes. It was particularly useful for the detection of newly generated granule cells that were difficult to identify with the autoradiographic method. This approach also allowed for the analysis of changes in the production of specific cell types under various experimental conditions. However, the immunohistochemical approach can also lead to false conclusions if potential technical problems are ignored (Nowakowski and Hayes, 2000, 2002; Rakic 2002). At this time there are no established criteria for the use of BrdU as a marker of neuronal birth date. It is therefore crucially important to establish basic standards for detecting the genesis of new cells and their identity.