Effects of excess thyroid hormone on cell death, cell proliferation, and new neuron incorporation in the adult zebra finch telencephalon.

Widespread telencephalic neuronal replacement occurs throughout life in birds. We explored the potential relationship between thyroxine (T4) and cell turnover in the adult male zebra finch. We found that many cells in the zebra finch brain, including long-projection neurons in the high vocal center (HVC), stained positively with an antibody to thyroid hormone receptors (TR). Labeling was generally weak in the ventricular zone (VZ) that gives rise to new neurons but some proliferative VZ cells and/or their progeny, identified by [3H]-thymidine labeling, co-labeled with anti-TR antibody. Acute T4 treatment dramatically increased the number of pyknotic and TUNEL-positive cells in HVC and other telencephalic regions. In contrast, degenerating cells were never observed in the archistriatum or sub-telencephalic regions, suggesting that excess T4 augments cell death selectively in regions that show naturally occurring neuronal turnover. VZ mitotic activity was not altered shortly after acute T4 treatment at a dosage that stimulated cell death, although [3H]-labeling intensity per cell was slightly reduced. Moreover, the incorporation rates for neurons formed shortly before or after acute hormone treatment were no different from control values. Chronic T4 treatment resulted in a reduction in the total number of HVC neurons. Thus, hyperthyroidism augmented neuronal death, which was not compensated for by neuronal replacement. Collectively, these results indicate that excess T4 affects adult neuronal turnover in birds, and raises the possibility that thyroxine plays an important role in the postnatal development of the avian brain and vocal behavior.

Targeted neuronal death affects neuronal replacement and vocal behavior in adult songbirds.

In the high vocal center (HVC) of adult songbirds, increases in spontaneous neuronal replacement correlate with song changes and with cell death. We experimentally induced death of specific HVC neuron types in adult male zebra finches using targeted photolysis. Induced death of a projection neuron type that normally turns over resulted in compensatory replacement of the same type. Induced death of the normally nonreplaced type did not stimulate their replacement. In juveniles, death of the latter type increased recruitment of the replaceable kind. We infer that neuronal death regulates the recruitment of replaceable neurons. Song deteriorated in some birds only after elimination of replaceable neurons. Behavioral deficits were transient and followed by variable degrees of recovery. This raises the possibility that induced neuronal replacement can restore a learned behavior.

Deafening alters neuron turnover within the telencephalic motor pathway for song control in adult zebra finches.

In the telencephalon of adult songbirds, projection neurons are lost and replaced within the efferent pathway controlling learned vocal behavior. We examined the potential role of auditory experience in regulating the addition and long-term survival of vocal control neurons in adult male zebra finches. Deafened and control birds were injected with the cell birth marker [(3)H]thymidine and then killed 1 or 4 months later. At the 1 month survival time, the number of [(3)H]-labeled neurons present in the high vocal center (HVC) was 70% lower in deafened birds compared with controls. This was true for all [(3)H]-labeled HVC neurons, as well as the subset that projected to the robust nucleus of the archistriatum. Over the next 3 months, two-thirds of the [(3)H]-labeled HVC neurons in control birds were lost, presumably through cell death. Surprisingly, deafened birds showed no loss over this interval. The total number of HVC neurons did not differ between control and deafened birds at either survival time. Nuclear diameters of [(3)H]-labeled HVC neurons decreased with cell age in both control and deafened birds, a process that may relate to the eventual death and replacement of these cells. These results suggest that experience influences the addition and also the longer-term fate of neurons formed in adulthood. We propose that auditory deprivation decreases the incorporation of new neurons and prolongs their life span. Alterations in the neuronal replacement cycle may relate to the gradual deterioration in song that occurs after deafening in adult zebra finches.

Fate of new neurons in adult canary high vocal center during the first 30 days after their formation.

Projection neurons are added to the high vocal center (HVC) of adult songbirds. Here we report on events associated with their initial arrival in HVC. Neurons formed in adult canaries were labeled with [(3)H]-thymidine and examined 8, 15, 22, and 31 days later. By 8 days, some [(3)H]-labeled cells with the nuclear profile of postmigratory neurons were already present in HVC but could not be retrogradely labeled by Fluoro-Gold injections in the robust nucleus of the archistriatum (RA); 7 days later, a few such cells could be backfilled from RA. Thus, new neurons may arrive in HVC as much as 1 week prior to establishing connections with RA. By 31 days, 43% of the [(3)H]-labeled neurons could be backfilled from RA. In no case were new neurons backfilled by tracer injections into Area X, suggesting that newly formed HVC cells do not establish a transient connection with this region. At all survival times, the somata of new neurons were often clustered tightly together with other HVC neurons that differed in age and projection. Between days 15 and 25 after their birth, half of the new HVC neurons disappeared. We conclude: (1) that neurons arrive in HVC earlier than previously thought, (2) that soon after their arrival they become part of cell clusters in HVC, and (3) that in addition to the previously described death of new neurons that occurs over a period of months, there is an early wave of death that occurs soon after new neurons adopt a postmigratory phenotype.

Birth, migration, incorporation, and death of vocal control neurons in adult songbirds.

Neurogenesis continues in the brain of adult birds. These cells are born in the ventricular zone of the lateral ventricles. Young neurons then migrate long distances guided, in part, by radial cell processes and become incorporated throughout most of the telencephalon. In songbirds, the high vocal center (HVC), which is important for the production of learned song, receives many of its neurons after hatching. HVC neurons which project to the robust nucleus of the archistriatum to form part of the efferent pathway for song production, and HVC interneurons continue to be added throughout life. In contrast, Area X-projecting HVC cells, thought to be part of a circuit necessary for song learning but not essential for adult song production, are only born in the embryo. New neurons in HVC of juvenile and adult birds replace older cells that die. There is a correlation between seasonal cell turnover rates (addition and loss) and testosterone levels in adult male canaries. Available evidence suggests that steroid hormones control the recruitment and/or survival of new HVC neurons, but not their production. The functions of neuronal replacement in adult birds remain unclear. However, rates of HVC neuron turnover are highest at times of year when canaries modify their songs. Replaceable HVC neurons may participate in the modification of perceptual memories or motor programs for song production. In contrast, permanent HVC neurons could hold long-lasting song-related information. The unexpected large-scale production of neurons in the adult brain holds important clues about brain function and, in particular, about the neural control of a learned behavior--birdsong.

Photoperiod regulation of neuron death in the adult canary.

The avian brain undergoes naturally occurring cell death and neuronal replacement in adulthood. Little is known about how neuron survival in adult birds is regulated. However, previous work suggests that this process is open to environmental control. We now report that a reduction in day length from spring-like to fall-like conditions can dramatically increase cell death in adult male canaries. Many of the dying cells are projection neurons in the motor pathway controlling song learning and production. Circulating levels of gonadal steroids were not correlated with photoperiod-induced changes in the magnitude of cell death. Our results suggest that neuronal death in adult male canaries is regulated by seasonal changes in photoperiod, and that this occurs independent of chronic changes in gonadal steroid hormone levels. Day length may serve as a predictive environmental cue to time cell death in accordance with seasonal reproduction.

Direct evidence for loss and replacement of projection neurons in adult canary brain.

Normally occurring projection neuron loss and replacement were quantified over a 6 month period in the pathway from the high vocal center (HVC) to the robust nucleus of the archistriatum (RA) in adult male canaries. Fluorescent latex microspheres were injected into RA in April--a procedure resulting in long-term retrograde labeling of RA-projecting HVC neurons. Labeled cell densities were then obtained 4 and 20 d later in April and 195 d later in October. We found that 41-49% of the RA-projecting HVC neurons present the previous April were no longer present in October. Fluorogold injections in RA 3 d prior to death in April and October retrogradely labeled similar overall densities of RA-projecting HVC neurons, indicating that cells lost over this 6 month period were replaced by new RA-projecting HVC neurons. Newer cells were larger than older cells, suggesting that an age-dependent reduction in size might precede death. Over the same time interval, no loss was observed for neurons projecting from the lateral magnocellular nucleus of the anterior neostriatum to RA. Thus, loss was specific to the input from HVC to RA. These findings raise the possibility that much if not all of the pathway from HVC to RA is replaced within a year. The time period examined encompasses the yearly transition from stable song to song learning in the canary (Nottebohm et al., 1986, 1987). A pronounced loss and replacement of neurons implicated in vocal control during this period may relate to the canary's ability to modify song in adulthood.

Production and survival of projection neurons in a forebrain vocal center of adult male canaries.

Neurons are produced in the adult canary telencephalon. Many of these cells are incorporated into the high vocal center (nucleus HVC), which participates in the control of learned song. In the present work, 3H-thymidine and fluorogold were employed to follow the differentiation and survival of HVC neurons born in adulthood. We found that many HVC neurons born in September grow long axons to the robust nucleus of the archistriatum (nucleus RA) and thus become part of the efferent pathway for song control. Many of these new neurons have already established their connections with RA by 30 d after their birth. By 240 d, 75-80% of the September-born HVC neurons project to RA. Most of these new projection neurons survive at least 8 months. The longevity of HVC neurons born in September suggests that these cells remain part of the vocal control circuit long enough to participate in the yearly renewal of the song repertoire.

Birth of projection neurons in adult avian brain may be related to perceptual or motor learning.

Projection neurons that form part of the motor pathway for song control continue to be produced and to replace older projection neurons in adult canaries and zebra finches. This is shown by combining [3H]thymidine, a cell birth marker, and fluorogold, a retrogradely transported tracer of neuronal connectivity. Species and seasonal comparisons suggest that this process is related to the acquisition of perceptual or motor memories. The ability of an adult brain to produce and replace projection neurons should influence our thinking on brain repair.

Cresyl violet: a red fluorescent Nissl stain.

Cresyl violet is widely used by neurobiologists to visualize Nissl substance in bright-field microscopy. Here we describe a method for using this dye as a red fluorescent Nissl stain. Unlike the bright-field staining technique, fluorescent cresyl is compatible with other fluorescent dyes and tracers, such as fluorescein, Fluoro-Gold and Fast Blue. The procedure requires only minor modifications of routine bright-field cresyl staining, the most significant being dilution of the stain. Thus, fluorescent red cresyl violet is simple to implement and may be of general use in fluorescence microscopy.

Genesis and death of vocal control neurons during sexual differentiation in the zebra finch.

Several song-related regions in the adult zebra finch brain have substantially more neurons in males than in females. Such differences appear to arise from sex differences in circulating steroids during early posthatch life. In the present study, developmental mechanisms involved in the production of sex differences are explored by examinations of the normal time course of posthatch neurogenesis and cell death in vocal control circuits. As a first step toward determining whether rates of neuron production may be different in males and females, tritiated thymidine, a marker of cell division, was administered to zebra finches at various times during the first month after hatching. Birds were sacrificed at 60 d. The number of cells formed after hatching and present at 60 d was then evaluated in 3 vocal control regions--HVc (hyperstriatum ventralis pars caudalis) and its 2 principal targets, RA (robust nucleus of the archistriatum) and Area X. Cell death was quantified by counts of normal and pyknotic, degenerating cells made in these nuclei in additional, untreated birds of both sexes at 5 d intervals from 5 to 45 d of age. The combined results of these experiments suggest that differential cell death is a major factor in the development of sex differences in the song control system and provide the first direct evidence for sex differences in cell death in the developing telencephalon. Although developmental time tables differ among the 3 brain areas examined, at specific ages significantly higher numbers of pyknotic cells were observed in HVc, RA, and presumptive Area X in females compared to males. Peak levels of cell death in RA occur 4-6 weeks after hatching. This is about 3 weeks after the onset of sex differences in steroid levels that, in turn, lead to differential organization of song system nuclei. This pattern of results suggests that designation for death and actual cell loss are temporally dissociated in this system. Neuron proliferation for HVc and Area X, but not RA, continues throughout the first 30 d after hatching, and a significant sex difference was found in the number of cells present in HVc at 60 d that were formed after hatching. Comparisons of the timing of cell death and cell incorporation suggest that this difference may be best accounted for by differential survival of neurons formed after hatching rather than differential rates of neuron production. Neither differential neurogenesis nor differential neuron death can fully account for the apparent extreme sexual dimorphism in the number of neurons in Area X.(ABSTRACT TRUNCATED AT 400 WORDS)

Song-related brain regions in the red-winged blackbird are affected by sex and season but not repertoire size.

Previous work in songbirds has delimited a neural system responsible for song production and control. Earlier studies have suggested that functional capacity in the song system may be related to the mass of the system in an animal's brain, and that adult plasticity in this neural system may be related to adult capacity for behavioral modification. We now test these hypotheses in adult red-winged blackbirds (Agelaius phoeniceus), a species in which song is produced primarily by males, new song types are added to the male's repertoire in adulthood, and there are substantial differences among males in song complexity. We find that the song system in males is much larger than in females. Song system nuclei become smaller in both sexes as the animals experience shorter days. We do not find any association between repertoire size and size of any of the song system structures examined. Thus, although sex differences in song may be related to differences between sexes in the mass of song system structures, individual differences in song do not appear to be directly related to mass within males. Seasonal change in song system structures in male redwings is consistent with there being a relation between adult plasticity in anatomy and in behavior; the large seasonal change in these structures in females suggests large seasonal changes in the function of these nuclei.