Proc. Natl. Acad. Sci. USAVol. 91, pp. 7844-7848, August 1994Neurobiology

Cell death and neuronal recruitment in the high vocal center ofadult male canaries are temporally related to changes in song

(neurogenesis/neuronal replacement/song larning/rejuvenatlon/onalt)

JOHN KIRN*, BARBARA O'LOUGHLINt, SUSAN KASPARIANt, AND FERNANDO NOTTEBOHMt*Department of Biology, Wesleyan University, Middletown, CT 06459-0170; and tRockefeller University Field Research Center, Tyrrel Road,Millbrook, NY 12545

Contributed by Fernando Nottebohm, April 8, 1994

ABSTRACT Adult male canaries modify their song everyyear. Most of these changes occur during late summer andearly fall, after the end of the breeding season, and in latewinter, immediately before the onset of the next breedingseason. The high vocal center (HVC) is an important nucleusin the brain pathway that controls this learned behavior. Newneurons continue to be added to the HVC of adult malecanaries, where they replace older neurons that have died. Thepresent report describes the monthly incidence of cell death andneuronal addition in the HVC of such birds. Different groupsof 1- to 2-year-old male canaries were treated with [3H]thymi-dine, a marker of cell birth, during each month of the year andkilled 27 days later. The ratio of 3H-labeled neurons to allneurons in the HVC showed seasonal peaks and troughs. Thisratio was highest in October and March. Peaks in the ratio ofpycnotic (dying) HVC cells to all neurons in HVC preceded thepeaks in the ratio of 3H-labeled neurons. We suggest thatseasonal peaks in cell loss and neuronal recruitment inHVC arerelated to endocrine changes and that all three play a role in theseaonality of song modification.

period of juvenile song acquisition (8-10). Production andreplacement of HVC neurons continues in adulthood (11).However, to our knowledge, there has been no description ofthe relative number of new neurons that adult male canariesadd to their HVC every month of the year. This informationwould tell us whether there is a temporal relation betweenchanges in the ratio ofnew to old neurons and changes in therate of appearance of new song syllable types. The presentstudy addresses this issue.Neurons added to the HVC of adult male canaries replace

older neurons that die (4, 12). HVC neuron loss has beenshown directly by labeling a subset of these cells and docu-menting its subsequent disappearance (4, 13, 14). However,we have not had, until now, a year-long series of monthlyobservations of cell death in HVC. The results we presenthere show that an increase in the fraction of cells that die inHVC precedes an increase in the fraction of newly formedneurons added to HVC. We suggest that seasonal peaks incell loss and in the recruitment of HVC neurons are relatedto the timing and extent of song modification.

The song of adult male canaries includes 20-40 syllabletypes. The stereotypy of these syllables shows marked sea-sonal changes. Song syllables are delivered in a very stereo-typed manner during the breeding season-e.g., March toJune. During this time, repetitions ofa same syllable look, ona sound-spectrograph, like carbon copies ofeach other. Songbecomes less frequent and syllables become less stereotypedafter the breeding season ends. In addition, most adult malecanaries stop singing altogether during a 2- to 3-week periodduring mid- or late summer. Instability in syllable structurepeaks in September, when a mean of two-thirds of all syllabletypes produced by each bird show poor stereotypy. Thesyllable instability seen in late summer is reminiscent of thatseen injuvenile canaries during the plastic song stage ofvocaldevelopment. A second shallow peak in syllable instabilityoccurs in February, when an average of one-seventh of allsyllable types produced by adult male canaries is unstable.Though adult male canaries can modify their song duringevery month of the year, loss, addition, and modification ofsong syllables is particularly evident during the periods ofsyllable instability (1).Song changes result, presumably, from changes in the

brain circuits that control this behavior. The most dramaticchange that occurs in the song circuits of adult canaries isneurogenesis and neuronal replacement in the high vocalcenter (HVC) (2-4). HVC is a telencephalic nucleus thatplays an important role in the acquisition and production oflearned song (5-7).

Earlier work showed that many HVC neurons are pro-duced late in ontogeny and, particularly, during the entire

MATERIALS AND METHODSAnimals. Adult males from our closebred colony ofBelgian

waterslager canaries were killed during every month of theyear, at ages ranging from 15 months to 28 months. Birdskilled at the beginning of the study (June) were the youngest;those killed at the end of the study were 1 year older. All ofthese birds had gone through one breeding season before theybecame part of our study. Birds were housed indoors, singlyor in small groups, in rooms maintained at 200C, under aphotoperiod that corresponded to that of New York State.Dry seeds, soaked seeds, and water were available ad libitumthroughout the year.

[3HJThymidine Treatment. [3H]Thymidine is incorporatedinto the nucleus of mitotic cells during the period of DNAsynthesis (S phase) that precedes actual cell division. In thisway it acts as a cell-birth marker (15). Each of 45 adult malecanaries received eight systemic i.m. (pectoral muscle) in-jections of 50 .Ci of [3H]thymidine [6.7 Ci/mmol; 1 Ci = 37GBq; New England Nuclear; =2.5 uCi/g (body weight)] at12-h intervals over a 4-day period and was killed by anoverdose of anesthetic 27 days after the last [3H]thymidineinjection. This treatment was given to 12 groups oftwo to sixbirds, one group for each month of the year. The 27-daysurvival time was chosen because earlier work showed thatit takes =20 days for neurons born in adult canary brain toreach their destination and acquire their adult phenotype (2,16, 17).

Histology and Quantification of [3H]Thymdine Results.Birds were perfused intracardially with phosphate-bufferedsaline (pH 7.4), followed by buffered 10%o (vol/vol) formalin.Thirty-six brains were embedded in paraffin, and eleven were

Abbreviation: HVC, high vocal center.

7844

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Proc. Natl. Acad. Sci. USA 91 (1994) 7845

embedded in polyethylene glycol (PEG). The paraffin-embedded brains were harvested during every month of theyear, but the PEG-embedded brains were obtained onlyduring February, March, and April. There was no systematicdifference in the ratio 3H-labeled neurons per 1000 HVCneurons associated with any one embedding method.Transverse sections were cut at a thickness of 6 gum,

processed for autoradiography as described (2), and stainedwith cresyl violet. Neuronal identification relied on thepattern of nuclear staining-cells with relatively large clearnuclei that had one or two darkly staining nucleoli wereconsidered neurons. This identification has been validated byusing ultrastructural criteria (2, 16) and also by using retro-gradely transported substances (11). However, we were notable to see the nucleoli of the most heavily labeled cells; inthese instances neuronal identification relied on a clearnucleoplasm and a nuclear size that was comparable to thatof other nearby lightly labeled or unlabeled neurons.HVC stands out in cresyl violet-stained sections because of

its characteristic cytoarchitecture and the light purple sheenof its neuropil. Whereas the dorsal boundary of HVC isformed by the floor of the lateral ventricle, the ventralboundary results from a rather abrupt transition in mean cellsize: neurons in HVC are larger and less densely spaced thanthose in the underlying neostriatum (5). There are times ofyear when the HVC boundaries determined in cresyl violet-stained material coincide very well with the boundariesobtained using retrograde markers injected into HVC targets.However, at other times boundaries shown by cresyl violetmay underestimate the true size of HVC (18). The presentstudy does not address this issue because cresyl violet wasthe only stain used. Due to this uncertainty, we have chosento present all our data as ratios-e.g., [3H]thymidine-labeledcells or pycnotic cells per 1000HVC neurons-rather than asabsolute numbers.A neuron was recognized as labeled when the number of

exposed silver grains overlying the nucleus was at least 10times that of the surrounding neuropil; in our material, thisusually corresponded to a minimum of three to six exposedsilver grains per nucleus. Two earlier studies failed to detectsystematic seasonal changes in the nuclear size, packingdensity, or number of HVC neurons in adult male canaries(12) or in HVC soma sizes (13), and so we did not modify ourcounts of labeled and unlabeled neurons with seasonal cor-

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rections. Two examples of 3H-labeled HVC neurons areshown in Fig. 1A.Counts of labeled HVC neurons were made in five or more

evenly spaced sections from left HVC. The right HVC wasused when its left counterpart was not available. The fewtimes that this happened the right-side results were wellwithin the distribution for the left side. Counts of total HVCneurons were made in three to five sections through thecenter of the HVC. We combined the counts of labeled andunlabeled neurons per unit area of HVC to arrive at anestimate of the number of labeled neurons per 1000 HVCneurons. The values obtained were divided by 4 to yield theratio of labeled neurons per 1000 HVC neurons per day of[3H]thymidine treatment. Counts of3H-labeled neurons weremade without knowing the month of year sampled.Counts of exposed silver grains over labeled neuronal

nuclei were compared in birds killed during months when ahigh (October) or a low (April, May, or June) proportion ofthe HVC neurons was labeled, to see whether these differ-ences in the ratio of labeled neurons could result fromdifferences in the duration ofthe S phase. All birds comparedhad gone through the same protocols of histological process-ing. Other things being equal, maximal label concentrationshould be inversely proportional to S-phase duration. In turn,the likelihood of a dividing cell becoming labeled by aninjection of [3H]thymidine would be directly proportional tothe duration of its S phase. If maximal labeling were com-parable at times of high and low recruitment, then this wouldsuggest that differences in the fraction ofneurons labeled didnot result from a difference in S-phase duration.Hstology and Quantification of Cel Death. Histological

treatment of the material used for counts of pycnotic cellswas the same as for counts of 3H-labeled neurons, and mostof our animals were used for both purposes. Pycnotic de-generating cells have darkly staining inclusions and usuallyshow a shrunken appearance (Fig. 1 B and C) that makes itdifficult to differentiate the nucleus from the cytoplasm andidentify the cell type (19-21). Therefore, we did not try todecide which of the pycnotic cells were glia and which wereneurons. Sometimes the darkly staining inclusions seemedfragmented; when such fragments were close enough tosuggest that they were part ofa single cell, they were countedas one (Fig. 1B). Pycnotic cell profiles were encountered lessfrequently and were more variable in number than 3H-labeledneurons. The relative paucity of pycnotic cell profiles may

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FIG. 2. Examples of spatial distribution of labeled HVC neurons.The boundaries shown were drawn from Nissl-stained material. Thesection from the bird killed in June (CTY-222) has many fewerlabeled cells than the section from the bird killed in October(CTY-251). However, in both cases the labeled cells occur through-out HVC. (Bar = 500 ,um.)

result from the brevity of this last stage of degeneration (22).It was, therefore, necessary to count pycnotic cells over alarge number of sections-typically, 15 per hemisphere(roughly, 10% of HVC). Our estimates of cell death are

expressed as the number of pycnotic cells per 1000 neurons.Since 3H-labeling and staining for pycnotic cells probablysample two time windows, it is inappropriate to compare theabsolute number of 3H-labeled or pycnotic cells, and we didnot do this.We culled two birds used in the [3H]thymidine series

because incomplete blood clearance during perfusion madethe scanning for pycnotic cells overly laborious. Erythro-cytes stain very darkly and when cut in cross section have adimension close to that of pycnotic cells. To make up forthese losses and the greater variability of the individualscores, an extra 10 birds were added to the monthly samplesof the [3H]thymidine series during months when otherwisethere would have been too small a number of birds to providea reliable mean number of pycnotic cells per 1000 HVCneurons. Counts of pycnotic cells were made without know-ing the month ofyear sampled. The ratio of pycnotic cells per1000 HVC neurons did not differ systematically betweenparaffin- and PEG-embedded material for months when bothmethods were used.The Spatial Distribution of 3H-Labeled and Pycnotic Cells.

3H-labeled neurons occurred throughout HVC in a fairlyhomogeneous manner. This distribution is shown in samplebirds that were killed in July and October (Fig. 2). Duringdevelopment, neurons also seem to be added throughoutHVC in a fairly homogeneous manner (9). Therefore, ourestimates of the ratio of 3H-labeled and pycnotic cells per1000 HVC neurons are not likely to have been influenced bythe exact position oftheHVC boundaries recognized with thecresyl violet stain.

RESULTS

[3H]thymidine-labeled neurons were found in HVC duringeach of the 12 months sampled but the ratio of 3H-labeledneurons to all HVC neurons was not the same during allmonths (ANOVA, F(11, 35) = 7.3; P = 0.0001). There weretwo significant peaks in this ratio, one in October and theother one in March (Fig. 3A). We compared the values foreach of these peaks with those of the month preceding andfollowing it by using the Student t test. The October values

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FIG. 3. (A) Number (mean ± SEM) of 3H-labeled HVC neuronsper 1000 HVC neurons in birds killed at different times of the year.Birds were killed 1 month after [3H]thymidine injection (see text).The number of birds in each monthly sample is indicated above theSEM bar. The letters on the horizontal axis are the first letter foreachmonth of the year, starting on the left with June (J). (B) Number(mean ± SEM) ofpycnotic cells per 1000HVC neurons. Other detailsare as in A. (C) Mean number of new syllable types that appeared inthe song of adult male canaries during their second year of life[modified from Nottebohm et al. (1)]. The shaded bars inking A-Cemphasize timing of the two peaks in neuronal recruitment and theirrelation to peaks in cell death and syllable acquisition.

were significantly higher than those of September (t(8) =

2.72; P = 0.026] and November [t(8) = 2.52; P = 0.036].Similarly, the March values were higher than those of Feb-ruary [t(9) = 2.43; P = 0.038] and April [t(9) = 5.22; P-=0.001]. In addition, the October peak was significantly largerthan the March peak [t(8) = 2.66; P = 0.029]. The Octoberratio was seven times higher than that seen in May.The mean and maximum number of exposed silver grains

per labeled neuronal nucleus did not differ between Octoberand April-June. The number of silver grains over the nucleusof labeled HVC neurons in October was 23.4 + 3.1 (mean +

SEM) and in April-June was 22.6 ± 8.6. This difference wasnot significant [t(7) = 0.19; P = 0.85]. Likewise, the numberof silver grains over the nucleus of neurons in the upper 10%oof the labeling distribution was comparable in October (60.0+ 7.8) and April-June (54.4 ± 16.7) [t(7) = 0.61; P = 0.56],

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7846 Neurobiology: Kim et al.

Proc. Natl. Acad. Sci. USA 91 (1994) 7847

suggesting that the duration of the S phase did not changeseasonally.

Pycnotic cells were found in HVC during each month oftheyear. In this case, too, an analysis of variance [ANOVA,F(11, 43) = 5.73; P = 0.0001] shows that monthly estimatesdiffered (Fig. 3B). During May, June, July, November, andDecember, pycnotic cells were extremely rare. During therest of the months, the occurrence of these cells was muchmore common. The highest ratios of pycnotic cells to HVCneurons were found in August and January, but these twopeaks did not differ significantly from each other [t(7) = 1.69;P = 0.13]. However, the January values differed significantlyfrom the values for December [t(6) = 5.15; P = 0.002] andFebruary [t(10) = 4.49; P = 0.001].The late summer peak in the ratio of pycnotic cells was

broader than the one in January, and therefore, for purposesof statistical comparisons, we pooled the values for Augustand September and compared those values to the pooledvalues of June and July and of October and November. Thebroad peak in the ratio ofpycnotic cells toHVC neurons seenin August and September was significantly higher than thevalues for the 2 months to either side [t(15) = 2.89; P = 0.01and t(17) = 2.71; P = 0.01, respectively]. As for 3H-labeledneurons, there was a 7-fold difference between the monthwith the highest ratio ofpycnotic cells (January) and that withthe lowest (May).

DISCUSSIONThe present results provide a 12-month survey of neuronalrecruitment and cell death in the HVC of adult male canaries.New HVC neurons and pycnotic cells were found during allmonths of the year. Thus, the present results suggest thatneurogenesis and cell death continue throughout the year inthe adult canary telencephalon.The proportion ofnew neurons recruited into HVC was not

the same during all months. Rather, this proportion peaked inOctober and again in March, with rates of0.74% and 0.5% perday of [3H]thymidine treatment, respectively (Fig. 3A). Thefall peak is consistent with prior work, in which [3H]thymi-dine injections made in October labeled substantially moreHVC neurons than injections in May (11). The more com-prehensive sampling method used in the present work, how-ever, has also uncovered a second smaller peak in theproportion of new HVC neurons in March.

Pycnotic cell counts, a measure of cell death, suggest thatHVC cell loss is not uniform throughout the year. As was truefor measures of neuronal recruitment, the ratio of degener-ating cells per 1000 HVC neurons exhibited two peaks. Thesepeaks occurred in August and September and in January.Both peaks in the incidence of pycnotic cells preceded peaksin the ratio of new HVC neurons by 2 months (Fig. 3B). Thetemporal relationship between waves of neuronal recruit-ment and increases in ratios of degenerating cells raises thepossibility that neuronal recruitment and cell death are caus-ally related.A prerequisite for the notion that there is a causal link

between peaks in neuronal recruitment and in the proportionof HVC cells dying is that the apparent seasonal differencesin recruitment and death are real and not the result ofphysiological processes that contaminated our cell counts.For example, pycnotic cell counts could be influenced byseasonal changes in the staining characteristics of dying cellsor by seasonal changes in the clearance time for degeneratingcellular debri. Our observations do not allow us to rule outthese possibilities. In the case of the [3H]thymidine results,seasonal differences in the duration of the S phase could leadto an appearance of seasonal differences in neuronal recruit-ment. However, histograms of the number of exposed silvergrains over the nucleus of labeled HVC neurons were com-

parable during October and April-June (data not shown),even though these two periods were associated with verydifferent ratios of3H-labeled neurons. We infer from this thatthe duration of the S phase of the cell cycle was comparableat these two times and that the seasonal differences in HVCneuronal recruitment are real. If so, then the apparent peaksin cell death are likely to be real too, part of the process ofneuronal replacement.

Pycnotic cell counts could reflect the death of eitherneurons or of nonneuronal cells. Previous counts of HVCneurons in 1- to 2-year-old male canaries sampled betweenOctober and May yielded constant numbers during this8-month period (12). This earlier study (12) and the researchreported here used birds of the same colony and the birdswere kept under very similar conditions. We infer that anyneuronal addition that occurred during those months in thepresent study was balanced by neuronal loss. Other work hasshown that a significant fraction of HVC neurons labeled inthe spring with fluorescent beads (13) or [3H]thymidine (14)disappears by early fall. The loss of such neurons is probablynot attributable to the label itself, because neurons similarlylabeled in the fall live much longer (12, 14). Thus, theseobservations suggest that many of our pycnotic cells weredead or dying neurons.We do not know whether the differences between months

in the ratio of3H-labeled cells per 1000HVC neurons resultedfrom differences in the production, migration, differentiation,survival of new neurons, or a combination ofthese variables.Because of this uncertainty, the neuronal recruitment valuescould have been shown for the month when the cells wereborn or for the month when the birds were killed. We chosethe latter approach (Fig. 3A) because it is the most descriptiveand makes the least number of assumptions. In addition, oursamples were taken at monthly intervals, and therefore, thetemporal relations seen have this degree of imprecision. Ourdata do not rule out the possibility that a peak in cell deathswas followed, less than a month later, by a peak in neuronalbirths.

Regardless of the nature of the linkage, if any, betweenHVC cell death and neuronal recruitment, the peaks of thesetwo events have an intriguing relation with periods ofmarkedvocal change reported in an earlier study (1, 23). This earlierstudy, too, used canaries of the same strain and age as thoseused in the present research. The earlier study described howthe song of adult male canaries becomes unstable during thesummer, after breeding ends, and then becomes again morestereotyped; a lesser period of unstable song occurs inmid-winter, before breeding starts (1, 23). Peaks in theemergence of new song syllables occur during these twoperiods of song instability and could be triggered by anincrease in HVC cell loss (Fig. 3C). The subsequent increasein the stereotypy of the new syllables could result from theaddition ofnew HVC neurons and a consequent rebuilding ofthis part of the song circuit. It is particularly remarkable thatthe proportional magnitude ofthe syllable additions (higher insummer than in winter) bears such a close relation to theproportional magnitude of the neuronal recruitment peaks.Though systemic injections of [3H]thymidine to adult male

canaries result in more labeled HVC neurons when given inOctober than when given in May, these injections do notresult in overall seasonal differences in the number of 3H-labeled neurons in the rest of the telencephalon (24). Weinfer, from this, that the seasonal patterns of neuronalrecruitment that we report here are particular to HVC and notto the whole brain. To this extent, the seasonal peaks in thedeath and recruitment of HVC cells may have evolved as anadaptation for seasonal changes in learned song.The preceding arguments link song learning to the replace-

ment of HVC neurons. Neuronal replacement, in turn, maybe regulated by hormones. The sharp increase in cell death

Neurobiology: Kim et al.

Proc. NatL. Acad. Sci. USA 91 (1994)

during August and January could result from the confluenceof low testosterone levels and low or falling estradiol levelsfound in the plasma of adult male canaries during these 2months (23). Cell death may, in turn, contribute to neuronalrecruitment by stimulating the division of neuronal stem cells(e.g., via diffusible factors) and by creating vacancies intowhich the new neurons can settle and differentiate. In addi-tion, the peaks in the ratio of new to old neurons seen duringOctober and March are probably encouraged by the increasein plasma testosterone levels during those two times of year(23). A companion paper (25) shows that testosterone pro-longs the life ofnewly generated HVC neurons that otherwisedisappear.

Thus, our results suggest that the yearly pattern of songchange found in adult male canaries is influenced by changinghormonal levels, changes in the proportion of dying HVCcells, and changes in the proportion of new to old HVCneurons. The ratio of new to old neurons may determine theextent of song modification.

We thank Yon Fishman for technical assistance. We thank Drs.Arturo Alvarez-Buylla, Barbara Finlay, and Ronald Oppenheim fortheir helpful comments. Marta E. Nottebohm provided skillfuleditorial help. Research reported in this publication was conductedwith the support of Public Health Service Grants MH18343 to F.N.and NS 29843 to J.K. In addition, this work benefited from thegenerous help of Mr. Howard Phipps, Mr. Herbert Singer, and theMary Flaggler Cary Charitable Trust.

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