Contributed by Fernando Nottebohm, April 8, 1994 ABSTRACT Adult male canaries modify their song every year. Most of these changes occur during late summer and early fall, after the end of the breeding season, and in late winter, immediately before the onset of the next breeding season. The high vocal center (HVC) is an important nucleus in the brain pathway that controls this learned behavior. New neurons continue to be added to the HVC of adult male canaries, where they replace older neurons that have died. The present report describes the monthly incidence ofcell death and neuronal addition in the HVC of such birds. Different groups

The number of high vocal center (HVC) neurons labeled in adult male caries by systemic Injections of [3H]thymidine depended on season and survival time. This was true for HVC neurons projecting to the robust nucleus of the archistriatum and for other HVC neurons that could not be retrogradely filled from the robust nucleus of the archistriatum. Birds Injected in October and killed 40 days later had twice as many labeled HVC neurons as birds injected in May and killed 40 days later. However, this difference became much larger (5 times) when the birds were allowed to survive for 4 months. Whereas more than half of the spring-born neurons disappeared between 40 days and 4 months, there was no reduction in the number of fall-born neurons present at the 4-month survival point. We infer that seasonal variables affect the life span of HVC neurons born in adulthood.It has been known for many years that the developing nervous system overproduces neurons. Many parts of the developing central nervous system retain only a fraction of the neurons originally produced (1-5). The conditions that determine this selective death are under intensive study (e.g., refs. 6-9). Neuronal death is also seen in some diseases ofthe adult human brain-e.g., Alzheimer and Parkinson diseases. Several studies (e.g., refs. 10-12) have looked at the factors or conditions that may prevent or delay neuronal death during development or in the adult diseased brain. There is hope that factors that prevent selective death during development may also rescue neurons that die during disease. But such studies need not be restricted to the developing brain. Neurogenesis and neuronal replacement occur spontaneously in a part of the song system of adult songbirds. This offers the opportunity to study neuronal death and replacement in a healthy adult vertebrate brain and in a system that controls a well-characterized behavior.Oscine songbirds acquire their song by reference to auditory information (13)(14)(15). The circuits involved in the acquisition and production of learned song have been described (16-19). One ofthe telencephalic nuclei, the high vocal center (HVC), necessary for the production of learned song (16,20,21), is of particular interest because it incorporates and replaces neurons in adulthood (22)(23)(24)(25)(26). More than half of the adult-formed neurons added to the HVC (26-28) grow an axon that reaches nucleus robustus archistriatalis (RA), on the efferent pathway for song production (16). The number of HVC neurons, including those that project to RA, ceases to increase in adulthood and neurogenesis in this system is part of a process of neuronal replacement (25,28,29).Earlier work (27) GBq; New England Nuclear). These birds received the PHIthymidine treatment at two times of the year, May (spring) or September (fall), and were divided into four groups as described in Table 1. All birds were weighed, anesthetized with 50 A1 of a dilution of Nembutal (Abbott; 10 mg/ml), and given FluoroGold [2% (wt/vol) solution in saline] microinj...

Tritiated thymidine has been widely used as a nuclear marker of cell birth. The true diameters and packing densities (nuclei/microns 3) of such radioactively labeled nuclei cannot be measured directly from tissue sections. Here we show that existing stereological corrections cannot be applied to data from radioactively labeled nuclei. We empirically measured the number of silver grains exposed by nuclei containing tritiated thymidine. The nuclei were separated from the photographic emulsion by known thicknesses of fixed, embedded avian telencephalon. The results of this experiment were used to develop an equation that estimates the number of silver grains exposed by a cell nucleus of any given diameter, containing a given amount of radioactive label, and located at any given distance from the photographic emulsion. The equation also allows one to calculate the probability that a label-containing nucleus will be correctly classified as labeled. Simulations of the equation revealed that not all label-containing nuclei are correctly classified by using commonly employed identification procedures and that larger nuclei are less likely to be correctly classified than smaller nuclei, given the same amount of label. The equation can be used to modify one class of existing stereological equations so as to be applicable to measurements of radioactively labeled nuclei. Finally, we discuss the assumptions and limitations of this modification.