A field study of seasonal neuronal incorporation ... - Wiley Online Library

A Field Study of Seasonal Neuronal Incorporation into the Song Control System of a Songbird That Lacks Adult Song Learning Anthony D. Tramontin,1 Eliot A. Brenowitz1,2 1

Department of Zoology, University of Washington, Box 351800, Seattle, Washington 98195-1800

2

Department of Psychology, University of Washington, Seattle, Washington 98195-1800

Received 14 January 1999; accepted 3 March 1999

ABSTRACT: Adult songbirds can incorporate new neurons into HVc, a telencephalic song control nucleus. Neuronal incorporation into HVc is greater in the fall than in the spring in adult canaries (open-ended song learners) and is temporally related to seasonal song modification. We used the western song sparrow, a species that does not modify its adult song, to test the hypothesis that neuronal incorporation into adult HVc is not seasonally variable in age-limited song learners. Wild song sparrows were captured during the fall and the spring, implanted with osmotic pumps containing [3H]thymidine, released onto their territories, and recaptured after 30 days. The density, proportion, and number of new HVc neurons were all significantly

The brains of adult birds continue to produce new neurons that can be incorporated into existing telencephalic circuits (reviewed in Alvarez-Buylla and Kirn, 1997). In the adult canary (Serinus canarius) forebrain, a neostriatal song control nucleus (HVc) can incorporate up to 1.5% of its neurons daily (Goldman and Nottebohm, 1983). More than 50% of these new neurons project to another premotor song control region, the robust nucleus of the archistriatum (RA) (Alvarez-Buylla et al., 1988, 1990; Nordeen and Nordeen, 1988). New projection neurons replace dying neurons in a process of ongoing Correspondence to: A. D. Tramontin Contract grant sponsor: NIH; contract grant numbers: MH53032, DC00395 Contract grant sponsor: NSF; contract grant number: DGE9616736AM02 © 1999 John Wiley & Sons, Inc. CCC 0022-3034/99/030316-11

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greater in the fall than in the spring. There was also a seasonal change in the incorporation of new neurons into the adjacent neostriatum that was less pronounced than the change in HVc. This is the first study of neuronal recruitment into the song control system of freely ranging wild songbirds. These results indicate that seasonal changes in HVc neuronal incorporation are not restricted to open-ended song learners. The functional significance of neuronal recruitment into HVc therefore remains elusive. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 316 –326, 1999

Keywords: adult neurogenesis; plasticity; song sparrow; testosterone; HVc

neuronal turnover (Kirn and Nottebohm, 1993). An important question regarding adult neuronal incorporation relates to the function of this form of neural plasticity. HVc is directly involved in song production and perception (Nottebohm et al., 1976; Brenowitz, 1991; Nordeen and Nordeen, 1992; Brenowitz et al., 1997). One hypothesis is that neuronal turnover in HVc might provide a neural substrate for plasticity of song production (Alvarez-Buylla, 1992; Alvarez-Buylla and Kirn, 1997). Adult canary songs undergo considerable seasonal changes in structure (Nottebohm et al., 1986). Canaries can retain existing song syllables, modify existing syllables, and produce novel syllables between breeding seasons (Nottebohm et al., 1986, 1987). Canaries are therefore described as open-ended song learners (Nottebohm and Nottebohm, 1978). Canary song modifications occur throughout the year, but are most

Seasonal Neuronal Incorporation into HVc

frequent during the nonbreeding season when song syllables are produced with less temporal and spectral stereotypy (Nottebohm et al., 1986). This peak period of song modification is closely associated with enhanced neuronal turnover in HVc (Kirn et al., 1994). Furthermore, the new neurons that are produced during this period live longer than those produced during the breeding season when song is stereotyped and stable (Nottebohm et al., 1994). These observations lead to the hypothesis that seasonal differences in neuronal incorporation underlie seasonal song learning (Nottebohm, 1989; Kirn et al., 1994). The strongest test of this hypothesis would consist of direct and specific manipulation of neuronal incorporation into HVc with subsequent behavioral assays of song learning. Until the technical challenges required for such a manipulation are solved, however, comparative studies of species that differ in the degree of adult song learning provide an alternative means of testing this hypothesis (Brenowitz, 1997). We therefore used a comparative approach to test whether HVc neuronal incorporation is seasonally variable in a songbird that does not modify its adult song repertoire. Unlike canaries, many songbird species are agelimited song learners. These birds learn songs only during their first year and then retain these songs throughout adult life. If seasonal changes in HVc neuronal incorporation are functionally related to seasonal song learning, one might predict that such seasonal changes would be less pronounced or absent in age-limited species. We tested this hypothesis in one such species, the western song sparrow (Melospiza melodia morphna), which has stable adult repertoires of six to 12 song types (Beecher et al., 1994b; Smith et al., 1997a). Song sparrows are seasonal breeders and show pronounced seasonal changes in the morphology of their song control systems (Smith et al., 1997a). Like canaries, song sparrows sing less stereotyped songs in the nonbreeding season. In contrast to canaries, however, song sparrows do not modify song syllables or add new syllables to their repertoires as adults (Marler and Peters, 1987; Cassidy, 1993; Smith et al., 1997a; M. D. Beecher, unpublished data). Here, we report that neuronal incorporation into the song sparrow HVc was greater in the fall than in the spring, as in canaries.

MATERIALS AND METHODS Field Procedures All protocols used in this experiment were approved by the University of Washington Animal Care Committee, the

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University of Washington Environmental Health and Safety Office, and the Washington State Office of Radiation Protection. We lured territorial adult male song sparrows into mist nets with tape-recorded playbacks of conspecific male song that simulated a territorial intrusion. We captured birds at the Charles L. Pack Experimental Forest in Washington in October 1996 (fall, n 5 5) and April 1997 (spring, n 5 6). Each bird was implanted subcutaneously on its back with an Alzet model 1007D micro-osmotic pump (length 5 17 mm; diameter 5 6 mm; filled weight 5 approximately 0.5 g) filled with 100 mCi of the cell birth marker [3H]thymidine (6.7 Ci/mmol; 1 Ci 5 37 GBq; New England Nuclear). The pumps each released their contents (100 mL) over 7 days at a rate of 0.62 mL/h. The pump rate was calculated by using the manufacturer’s calibration equation to adjust for song sparrow body temperature (41°C) and extracellular fluid osmolality (350 mmol/kg). This pump rate has been validated previously in this species (Goldstein and Rothschild, 1993). To determine whether implantation was detrimental to the sparrows’ health, we measured each bird’s body mass and estimated fat depots in the furcular fossa and abdominal cavity using the method of Wingfield and Farner (1978). Each bird was marked with a unique combination of colored leg bands before being released back onto his territory. At 30 days postimplant, each male sparrow was recaptured. We collected a small blood sample for hormone analysis, reweighed each bird, rechecked fat depots, and measured the length of the cloacal protuberance (an androgen-sensitive secondary sex organ). We also determined whether each bird still had a pump implanted. In the fall, two of five birds still had an implant after 30 days. In the spring, four of six birds retained the implants until recapture. In a different experiment using the same micro-osmotic pump implantation technique in wild song sparrows, we recaptured 43 birds (20 during the spring and 23 during the fall) at 14 days postimplantation. In that study, all of the birds retained their pumps for this period (Soma et al., 1998). We are therefore confident that all birds in the present study retained their pumps for at least the 7 days required for delivery of their entire contents.

Histology and Morphometry Each male was brought back to the laboratory on the day of recapture, deeply anesthetized with methoxyflurane (Metofane; Pitman-Moore), and perfused through the heart with avian saline followed by 4% paraformaldehyde. The testes were removed and weighed. Brains were postfixed for 18 h, rinsed in water for 8 h, dehydrated through a graded ethanol series, cleared in methyl salicylate, and embedded in paraffin. Then, 12- and 6-mm transverse sections were cut and mounted with an interval of 48 mm between sections. We labeled HVc in 12-mm sections with thionin and in 6-mm sections with Hu immunocytochemistry (below). We projected an image of each mounted section onto paper at a final magnification of 346. We traced the outline of each brain nucleus profile, scanned the tracings into a microcom-

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puter, and calculated the area of each nucleus profile using NIH Image (Ver. 1.57; Wayne Rasband, National Institutes of Health). We estimated the volume of each nucleus using the formula for a cone frustum over each measured area (Smith et al., 1997c).

Combined Hu Immunocytochemistry and [3H]Thymidine Autoradiography To identify new neurons in HVc, we used [3H]thymidine autoradiography in conjunction with monoclonal antibody 16A11 (Monoclonal Antibody Facility, University of Oregon) directed against the Hu family of vertebrate neuronal proteins (Marusich et al., 1994; Barami et al., 1995). Hu is a neuron-specific RNA-binding protein and is homologous to the elav gene from Drosophila melanogaster (Robinow and White, 1988). Hu is constitutively and ubiquitously expressed in the adult avian brain (Barami et al., 1995). Slides were deparaffinized, rinsed in Tris-HCl buffer, and treated with pronase (7 U/mL) for 4 min. Following three washes in Tris and three washes in phosphate-buffered saline (PBS), the tissue was preblocked in 10% normal horse serum for 30 min and then incubated with the Hu antibody (6.25 mg/mL) overnight in a humidified chamber at 4°C. Following five PBS rinses, Hu binding was visualized with avidin– biotin amplification (Vectastain Elite; Vector Labs) and diaminobenzidine chromagen. Negative control slides were prepared for each immunostained brain by omitting either the primary or the secondary antibody. These slides showed no staining of cells or neuropil. Slides were rinsed in five changes of PBS and five changes of distilled water, dried at room temperature for 24 h, and then dipped into Kodak NTB-3 nuclear track emulsion. After 5 h of drying, slides were packed into desiccated, light-proof boxes at 4°C for 60 days, and then developed in Kodak D19 developer.

Neuronal Attributes Throughout HVc we sampled the overall density and number of neurons, cross-sectional somal area, and neuronal nucleus diameter in every other mounted 6-mm section (sampling interval between sections 5 96 mm). We used a random, systematic sampling protocol that has been previously described and validated (Tramontin et al., 1998). Neuronal attributes were not measured in one spring bird because of poor histology. The use of [3H]thymidine required thin histological sections (6 mm) so that b particles could penetrate the tissue and expose the overlying emulsion. With such thin sections there was a possibility of splitting neuronal nuclei between sections, which could bias cell counts (West, 1993; Coggeshall and Lekan, 1996). To estimate neuron density, we counted neuronal nuclei in HVc and used Konigsmark’s (1970) formula to correct for nucleus splitting: N/n 5 t/(t 1 2a)

Figure 1 Combined Hu immunocytochemistry and [3H]thymidine autoradiography. Note exposed silver grains over the nucleus of a Hu-immunoreactive HVc neuron. Scale bar 5 20 mm. where N is the number of nuclei present, n is the number of nuclei counted, t is the section thickness in micrometers, and a is the square root of r22(k/2)2. Here, r is the nucleus radius, and k is the diameter of the uncounted (“invisible”) fragments of nuclei. We set k equal to 1 mm, which equaled the smallest nucleus fragment encountered in this study. Konigsmark-corrected neuron counts were divided by the volume of the tissue sampled to obtain neuronal density. We estimated HVc neuron number by multiplying neuron density by total HVc volume. These calculations are all based on several assumptions, but we were more interested in relative seasonal differences than in absolute numbers of HVc neurons (Saper, 1996; Guillery and Herrup, 1997). Any biases introduced by these assumptions should equally affect counts from both seasons (see below). We sampled a minimum of 150 HVc neurons in each brain. This sample size is sufficient to encompass the entire range of variability in neuron density and somal area in HVc (Tramontin et al., 1998). All measurements were made blind to the treatment of each bird. 3 H-labeled neurons were infrequent in the song sparrow telencephalon. We therefore used a modified random, systematic protocol to estimate the density of these neurons in HVc and in the adjacent neostriatum. After a random start, we scanned the entire area of HVc in every other mounted section and counted 3H-labeled HVc neurons (sampling interval between sections 5 96 mm; scanning magnification 5 3125). These counts were Konigsmark-corrected as described above to estimate 3H-labeled neuron density and number. In each section, we also sampled an area of neostriatum not involved with song control that was ventral to and equal in size to HVc. An immunoreactive neuron was considered labeled if it had at least four silver grains situated over its nucleus and exceeded a 320 background criterion for labeling (Fig. 1). Background labeling over the neuropil was determined in every visual microscope field

Seasonal Neuronal Incorporation into HVc Table 1

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Reproductive Characteristics (Mean 6 S.E.M.)

Plasma testosterone (ng/mL) Paired testes mass (g) Cloacal protuberance length (mm)

Fall (n 5 5)

Spring (n 5 6)

U*

p*

0.07 6 0.00 0.01 6 0.01 2.20 6 0.30

0.63 6 0.32 0.36 6 0.02 7.97 6 0.29

15 15 15

.004 .004 .004

†

* Mann–Whitney U (two-tailed). † Plasma testosterone was not detectable in any fall samples. All samples are reported as 0.07 ng/mL, which was the lower limit of detectability for this assay (see Materials and Methods).

that contained a labeled neuron. This protocol resulted in sample sizes ranging from 26 to 96 new HVc neurons and 29 to 51 new neostriatal neurons per bird. In each bird, neuronal recruitment into HVc was expressed in terms of density (3H-labeled neurons/HVc volume), proportion (number of 3H-labeled neurons/1000 HVc neurons), or number (3H-labeled neuron density 3 total HVc volume). Previous reports of HVc neuronal recruitment have used one or more of these three measures. There are distinct advantages and disadvantages to each of the three methods of expressing neuronal recruitment (Kirn and DeVoogd, 1989; Burek et al., 1997). Differences in density and proportion are sensitive measures of neuronal recruitment. Ratios, however, can change because of changes in the numerator or changes in the denominator. We have therefore reported all three recruitment measures in this study. The size of 3H-labeled neuronal nuclei (our unit of count) did not differ between groups, so it is unlikely that splitting errors introduced an asymmetric bias to our data (Saper, 1996; Guillery and Herrup, 1997). Furthermore, as mentioned above, we were most interested in relative seasonal differences in these measures.

RESULTS Behavior, Plasma Testosterone Concentrations, and Body Measurements Song sparrows are territorial throughout the year (Wingfield and Hahn, 1994). Micro-osmotic pump

Hormone Assay We collected 300 mL of whole blood from each male by alar venepuncture into heparinized capillary tubes. The blood was centrifuged and the plasma was removed and stored at 220°C until assay. We measured plasma testosterone (T) using the Coat-A-Count Total Testosterone radioimmunoassay kit (Diagnostic Products Corp.). The minimum detectable plasma T concentration was 0.07 ng/mL. Samples with undetectable levels of steroid were treated as having concentrations at this detection limit for statistical analysis.

Statistical Analysis Comparisons between seasons were performed with t tests. When parametric assumptions were violated, we used nonparametric Mann–Whitney U or Wilcoxon signed ranks tests. HVc volume comparisons were performed with repeated measures two-way analyses of variance (ANOVAs) (between subjects 5 season; within subjects 5 histological label). The a level for all statistical comparisons was .05 (two-tailed).

Figure 2 Seasonal differences in 3H-labeled HVc neuron density. (A) The density of 3H-labeled neurons (mean 6 S.E.M.) in HVc (solid bars) was significantly greater in the fall than in the spring. There was also a significant seasonal difference in the neostriatum adjacent to HVc (hatched bars), but this difference was less pronounced. (B) The normalized density of 3H-labeled neurons (mean 6 S.E.M.) in HVc was significantly greater in the fall than in the spring. 3H-Labeled neuron density measures were normalized by subtracting neostriatal density from HVc density. Sample size 5 5 in both seasons. Asterisks indicate significant statistical differences between seasons.

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Figure 3 Seasonal differences in the proportion and number of 3H-labeled HVc neurons. (A) The proportion of 3 H-labeled neurons (mean 6 S.E.M.) in HVc was significantly greater in the fall than in the spring. (B) The estimated number of 3H-labeled neurons (mean 6 S.E.M.) in HVc was also significantly greater in the fall than in the spring. Sample size 5 5 in both seasons. Asterisks indicate significant statistical differences between seasons.

implantation had no effect on territorial status or song behavior. At the time of recapture, all males were aggressively defending territories. In the fall, four of five birds sang in response to playback just prior to recapture, and all six birds sang just prior to recapture in the spring. The song sparrows were in different reproductive states in the spring and fall. Plasma testosterone concentration, paired testes mass, and cloacal protuberance length were all greater in the spring breeding season (Table 1). Micro-osmotic pump implantation had no effect on body mass (t 5 20.09, df 5 10, p 5 .929: paired t test) or fat score (W 5 25, p 5 .688: Wilcoxon signed rank test), suggesting that all the birds were in good physical condition.

Incorporation of New Neurons into HVc The incorporation of new neurons into HVc changed seasonally and was greater in the fall than during the spring. The density of 3H-labeled neurons in HVc was

significantly greater during the fall (F) than during the spring (S) (F:S 5 2.92, t 5 3.65, df 5 8, p 5 .006: t test) [Figs. 1 and 2(A)]. There was also a seasonal difference in the density of 3H-labeled neurons in the adjacent neostriatum (F:S 5 1.56, t 5 2.47, df 5 8, p 5 .038: t test), but this was less pronounced than the difference in HVc [Fig. 2(A)]. To examine this more closely, we subtracted the neostriatal density measure from the HVc density measure [Fig. 2(B)]. After normalizing the density of 3H-labeled HVc neurons in this way, we still found a significant effect of season (t 5 2.80, df 5 8, p 5 .023: t test). The proportion of 3H-labeled neurons in HVc changed seasonally, as did the estimated number of 3 H-labeled HVc neurons (Fig. 3). The proportion of new HVc neurons was greater in the fall than in the spring (F:S 5 2.51, t 5 3.15, df 5 8, p 5 .014: t test) [Fig. 3(A)]. Proportional data can reflect a change in either neuronal incorporation (numerator) or overall neuron number (denominator). We therefore estimated the number of new neurons incorporated into HVc during both seasons (see Materials and Methods). This estimated number of new HVc neurons was significantly greater in the fall than in the spring (F:S 5 1.65, t 5 2.38, df 5 8, p 5 .044: t test) [Fig. 3(B)]. The somal area of new HVc neurons differed between seasons (F:S 5 0.73) and was greater in the spring. The mean somal area of 3H-labeled neurons was 82.13 6 2.48 mm2 [mean 6 standard error of the mean (S.E.M.)] in the fall and 112.23 6 9.87 mm2 in the spring (t 5 22.96, df 5 8, p 5 .018: t test). The mean nucleus diameter of 3H-labeled neurons did not change seasonally (7.70 6 0.33 mm in the fall and 8.88 6 0.59 mm in the spring: F:S 5 0.87, t 5 21.76, df 5 8, p 5 .117: t test). Figure 4 shows that the

Figure 4 Autoradiography grain distribution histograms. The distributions of the number of silver grains over 3Hlabeled HVc neurons were similar in the fall (solid bars) and spring (hatched bars) groups. The mean number of silver grains over new HVc neurons did not differ between seasons (p 5 .768). Sample size 5 5 in both seasons.


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tissue than in Hu-stained tissue during the fall but slightly larger in the spring. Several neuronal attributes of HVc (3H-labeled and unlabeled neurons) changed seasonally (Table 2). Neuronal soma area and nucleus diameter were significantly larger in the spring (F:S 5 0.62 and 0.84, respectively) (Table 2 and Fig. 6). Neuron density was lower in the spring (F:S 5 1.17), while neuron number was significantly greater in the spring (F:S 5 0.68) (Table 2). Figure 5 Seasonal differences in HVc volume. HVc volume (mean 6 S.E.M.) differed significantly between seasons. HVc volumes were measured in 12-mm thioninstained sections (solid bars) or in 6-mm Hu antibody– labeled sections (hatched bars). In both seasons, the two staining methods resulted in similar HVc volume estimates. Sample sizes 5 5 (fall) and 6 (spring). Asterisks indicate significant statistical differences between seasons.

number of exposed silver grains over 3H-labeled HVc neurons was similar between seasons. The mean number of silver grains over neuronal nuclei did not change seasonally (6.56 6 0.46 in the fall and 6.38 6 0.38 in the spring: t 5 0.31, df 5 8, p 5 .768: t test). In addition, the degree of neuronal 3H-labeling (nuclear labeling/background labeling) did not differ significantly between seasons (250.85 6 80.36 in the fall, and 121.04 6 32.48 in the spring: t 5 1.498, df 5 8, p 5 .173: t test).

General Neural Attributes of HVc The overall volume of HVc differed significantly between seasons and was smaller in the fall than in the spring [F:S 5 0.56, F(1,21) 5 17.76, p 5 .002: repeated measures ANOVA] (Fig. 5). The measured volume of HVc was similar in thionin-stained and Hu-immunolabeled sections [F(1,21) 5 0.66, p 5 .436: repeated measures ANOVA]. There was a significant interaction between season and labeling method [F(1,21) 5 7.61, p 5 .022: repeated measures ANOVA]. This interaction is evident in Figure 5, where HVc volume is slightly smaller in Nissl-stained Table 2

DISCUSSION We investigated seasonal adult neuronal incorporation in wild songbirds that lived freely in their natural habitat. To investigate neuronal recruitment into the hippocampus, Barnea and Nottebohm (1994) similarly released black-capped chickadees (Parus atricapillus) in the field after single injections of [3H]thymidine. The micro-osmotic pumps allowed us to deliver [3H]thymidine at a constant rate over several days (Brown et al., 1993). This procedure provides new opportunities for field experiments on neuronal plasticity. In nature, birds are exposed to the full range of environmental and social cues that stimulate seasonal physiological changes and naturally occurring brain changes. Field investigations will be crucial in understanding how the brain responds to environmental cues such as day length, food availability, mate availability, and temperature. Future field studies will yield unique insights into the functional relevance of plasticity in the brain.

Incorporation of New Neurons into HVc The recruitment of new neurons into the adult HVc changed seasonally in wild male song sparrows. Just as in canaries, the density, proportion, and number of 3 H-labeled HVc neurons were all greater in sparrows captured during the fall (Alvarez-Buylla et al., 1990; Kirn et al., 1994). The magnitude of the seasonal difference, however, appeared to be smaller in song

HVc Neuronal Attributes (Mean 6 S.E.M.)

Neuronal somal area (mm ) Neuronal nucleus diameter (mm) Neuron density (31000 cells/mm3)† Neuron number (31000)† 2

Fall (n 5 5)

Spring (n 5 5)

t*

p*

63.95 6 2.72 6.46 6 0.14 235.04 6 12.85 126.09 6 10.91

103.84 6 5.39 7.70 6 0.26 201.29 6 5.60 186.71 6 11.93

26.61 24.20 2.40 23.75

,.001 .003 .043 .006

* Student unpaired t test (two-tailed). † Estimated from Konigsmark-corrected neuron counts.

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Figure 6 HVc neurons immunolabeled with Hu during the fall and the spring. HVc neurons were larger with larger nuclei during the spring. Scale bar 5 40 mm.

sparrows than in canaries. The F:S ratio of 3H-labeled neurons/1000 HVc neurons was 2.51 in song sparrows [Fig. 3(A)] and was approximately 4.25 in canaries killed at comparable times of year by Kirn et al. (1994). These data are consistent with the hypothesis that open-ended and age-limited song learners differ in the absolute amount of seasonal neuronal incorporation into HVc. One should note, however, that our study used very different methods from those of Kirn et al. (1994). A careful investigation of this hypothesis will require that open-ended and age-limited songlearning species be subjected to the same method and time course of [3H]thymidine treatment. The hypothesis that the magnitude of seasonal HVc neuronal incorporation differs between openended and age-limited learners should be tested with comparative studies using carefully chosen species to take advantage of the rich behavioral diversity among songbirds. Such studies may elucidate the functional relevance of neuronal incorporation into the adult song control system. Song mimics such as European starlings (Sturnus vulgaris) that learn new conspecific and heterospecific vocalizations as adults (AdretHausberger et al., 1990; Eens et al., 1992; Eens and Pinxten, 1993) might be predicted to show higher ongoing levels of HVc neuronal incorporation and/or turnover than canaries if the development of new adult songs requires the formation of new synapses (Alvarez-Buylla et al., 1990; Alvarez-Buylla, 1992). Song learning appears to decrease with age in adult starlings (Eens et al., 1992). Perhaps an age-dependent decrease in adult neuronal incorporation into HVc underlies the age-dependent decrease in song

learning. Another interesting species for study is the spotted towhee (Pipilo maculatus). This species shows the greatest seasonal plasticity in the song control nuclei of any bird studied thus far, but is an age-limited song learner. Towhees, unlike song sparrows, do not sing during the nonbreeding season, and therefore do not show the same seasonal changes in song stereotypy as seen in canaries and song sparrows (Davis, 1958; Ewert, 1978). Studies of HVc neuronal incorporation in towhees might provide clues as to whether adult neuronal incorporation is in fact related to seasonal plasticity of song structure. The seasonal differences in neuronal recruitment into the song sparrow HVc were not due to differences in [3H]thymidine incorporation or neuronal sampling factors. We detected no seasonal difference in the grain distribution histograms (Fig. 4) or the mean number of exposed silver grains over labeled HVc neurons. These results, coupled with constant [3H]thymidine release from the osmotic pumps, make it unlikely that observed differences in neuronal recruitment resulted from seasonal differences in [3H]thymidine incorporation. The mean nuclear diameter of HVc neurons changed seasonally and was smaller in the fall than in the spring. The mean nuclear diameter of 3H-labeled HVc neurons, however, was not significantly different between seasons and so probably did not affect our 3H-labeled neuron counts. Furthermore, the F:S ratio of nuclear diameter was not large enough to significantly affect our counts of 3 H-labeled cells (Clark et al., 1990; Nottebohm et al., 1994). Consistent with these observations, the degree of neuronal 3H-labeling (neuronal labeling/back-


ground labeling) did not differ seasonally and was well above the minimum labeling criterion (320 background) in both seasons. Seasonal differences in neuronal recruitment could have arisen from seasonal differences in neuronal proliferation, migration, differentiation, survival, or a combination of these factors (Kirn et al., 1994). We cannot distinguish among these alternatives. In songbirds, seasonal neuronal recruitment has not been reported in brain structures other than HVc and the hippocampal complex (Alvarez-Buylla et al., 1994; Barnea and Nottebohm, 1994). Our results, however, suggest that seasonal patterns of neuronal recruitment do exist outside these regions in song sparrows. In addition to the seasonal differences in HVc, we observed a significant but less pronounced seasonal change in neuronal incorporation in the neostriatum outside of HVc (Fig. 2). In this region of the neostriatum, 3H-labeled neurons were more densely spaced in the fall than in the spring. The functional significance of seasonally variable neuron incorporation outside HVc is unclear. This result, however, emphasizes the importance of measuring neuronal recruitment in brain regions that are not involved in song control.

General Neuronal Attributes of HVc HVc volume was nearly 100% larger in the spring than in the fall. The estimated number of HVc neurons was greater during the spring. This replicates the results of Smith et al. (1997a) and supports the hypothesis that neuron number changes seasonally in HVc (see also Gahr, 1990; Brenowitz et al., 1991; Johnson and Bottjer, 1993; Bernard and Ball, 1995; Smith et al., 1997b; Soma et al., 1997). Seasonal patterns of neuronal recruitment may contribute to seasonal changes in HVc volume and neuron number (Nottebohm, 1989). The vernal increase in neuron number in HVc could be accomplished by a downregulation of cell death, an up-regulation of cell incorporation, recruitment of existing neurons from the surrounding neostriatum, or a combination of these mechanisms. It will be important to investigate neuronal incorporation and death in the song sparrow HVc during the early spring when HVc grows, and during the late summer when HVc regresses. HVc neuron number is low in the fall despite enhanced neuronal recruitment during this season. For this to occur, cell death must keep pace with neuronal incorporation in this season. To test this hypothesis, it will be necessary to measure seasonal neuronal cell death in song sparrows and other species. Kirn et al. (1994) found seasonal patterns of cell death in the

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canary HVc, but could not discriminate between pycnotic neurons and glia in their tissue. Seasonal patterns of neuronal death might be elucidated by using Hu immunocytochemistry in concert with the TUNEL (TdT-mediated dUTP Nick End Labeling) assay for dying cells.

Plasma Testosterone Concentrations Gonadal steroids are thought to influence adult neuronal turnover in HVc. Exogenous testosterone (T) and estrogens can increase the recruitment and/or life span of new HVc neurons in adult female canaries (Rasika et al., 1994; Hidalgo et al., 1995). Cell death in HVc is preceded by a decrease in plasma T in male canaries (Kirn et al., 1994; Alvarez-Buylla and Kirn, 1997). In song sparrows the concentration of plasma T differed seasonally and was lower in the fall when neuronal recruitment was higher. This result is consistent with data from canaries (Alvarez-Buylla and Kirn, 1997). Alvarez-Buylla and Kirn (1997) hypothesized that cell death induced by decreasing T levels in fall might create vacancies in HVc that are subsequently filled when rising T provides new cells with trophic support. The fall sparrows in our study received [3H]thymidine in October and showed increased neuronal recruitment into HVc, although plasma T does not begin to increase in this species until late February (Wingfield, 1994; Tramontin et al., unpublished data). Perhaps neurons are incorporated at a high rate in the fall owing to vacancies, but also turn over at a high rate in the absence of T. This would explain how the song sparrow HVc maintains fewer total neurons in the fall despite increased neuronal incorporation. As T levels rise in preparation for breeding, new neurons may be maintained until vacancies are filled, resulting in the vernal increase in the volume of HVc. The annual cycle of plasma T differs between canaries and song sparrows. It is unclear whether the pattern of steroidal control of neuronal recruitment also differs between species. The annual cycle of plasma T shows two temporal peaks in canaries (Nottebohm et al., 1987), accompanied by two waves of neuronal incorporation (Kirn et al., 1994). AlvarezBuylla and Kirn (1997) noted that canary HVc neurons born in the fall appear to survive despite the ensuing plasma T decrease that occurs during January in that species. This might suggest that T exerts different effects depending on the time of year when a neuron is born. Perhaps other factors act in concert with T to control neuronal recruitment and/or survival (Alvarez-Buylla and Kirn, 1997). Song sparrows undergo only one major seasonal

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peak in plasma T (Wingfield and Hahn, 1994). T begins to rise in late winter and HVc begins to grow at this time (Wingfield, 1994; Smith et al., 1997a; Tramontin et al., unpublished data). T reaches its maximum level in early spring and then falls to an intermediate level after the first clutch of eggs is laid (Wingfield and Hahn, 1994). If hormonal mechanisms underlying neuronal turnover are similar in these two species, it will be interesting to investigate neuronal death and incorporation into HVc during the late winter, early spring, and late summer, when this nucleus is undergoing pronounced growth and regression. Comparative studies might more clearly elucidate hormonal mechanisms underlying cell birth, survival, and death.

Conclusions Our data strongly suggest that seasonal changes in HVc neuronal incorporation are not restricted to openended song learners. Song sparrows do not modify or add song syllables seasonally. Like canaries, however, song sparrows sing with less stereotypy during the nonbreeding season (Smith et al., 1997a). Perhaps seasonal changes in HVc neuronal recruitment are more closely correlated with song stereotypy changes than to song modifications or to song learning. These changes in song stereotypy may be a precursor to the development of new adult songs that is typical of open-ended song learning. From this perspective, seasonal changes in HVc neuronal incorporation may enable open-ended song development, but these neural changes may occur in birds without resulting in the seasonal development of new song patterns by adults (Brenowitz et al., 1991). Seasonal differences in neuronal recruitment may not be related to song production, but instead may be related to song perception (Alvarez-Buylla et al., 1990; Alvarez-Buylla and Kirn, 1997). There is behavioral and electrophysiological evidence that HVc is involved in song perception (Brenowitz, 1991; Margoliash, 1997). Furthermore, adult song sparrows learn to recognize and discriminate among the songs of several territorial neighbors each year (Stoddard et al., 1990, 1991, 1992; Beecher et al., 1994a). Currently, however, there is little evidence that song perception changes seasonally (cf. Cynx and Nottebohm, 1992). At present, the functional significance of neuronal recruitment in HVc remains an open question. The authors thank Ed Rubel for providing facilities and advice throughout this project. Glen MacDonald, Dale Cunningham, Judy Debel, and Karin Lent assisted with tissue

processing and analysis. Kathy Nordeen provided advice on the use of mAB 16A11. She, Kiran Soma, and two anonymous reviewers provided helpful comments on the manuscript. This work was supported by NIH MH53032 to EAB and NIH DC00395 to E. W. Rubel. ADT is supported by NSF DGE9616736AM02.

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