Proc. Nati. Acad. Sci. USA Vol. 91, pp. 7854-7858, August 1994 Neurobiology Testosterone increases the recruitment and/or survival of new high vocal center neurons in adult female canaries (neurogenesis/neuronal replacement/song system/gonadal steroids/plastIcIty) S. RASIKA*t, F. NOTTEBOHM*, AND A. ALVAREZ-BUYLLAt *Laboratories of Animal Behavior and tDevelopmental Neurobiology, Rockefeller University, New York, NY 10021 Contributed by F. Nottebohm, May 3, 1994

ABSTRACT New neurons are added to the high vocal stable (11). This work also reported that the size of HVC center (HVC) of adult male and female canaries. Exogenous grows dramatically in these birds under the influence of testosterone induces a marked increase in HVC size in adult testosterone over a period of 30 days, although the cellular female canaries, though the mechanisms responsible for this mechanisms behind this growth were not determined. Thus, increase remain unknown. To understand the mechanisms, we these observations on adult male and female canaries suggest analyzed the effects of testosterone on neuronal recruitment in that testosterone plays an important role in the recruitment of the female HVC. Intact adult female canaries received Silastic HVC neurons generated in adulthood. implants that were empty or filled with testosterone. Birds in Here we describe experiments that suggest that testoster- the short-survival group received the Silastic implant, followed one influences the recruitment and/or survival of newly by a single injection of [3H]thymidine 2 days later, and were generated neurons in HVC. Testosterone does not seem to killed on the following day. Birds in the long-survival group influence the number of dividing VZ cells, either above the were injected once a day for 5 days with [3Hlthymidine and HVC or elsewhere (see ref. 12). In addition, we will show that received the Silastic implant 20 and 40 days later. These birds testosterone also affects the nuclear and soma size of HVC were killed 60 days after the first injection of [3Hlthymidine. neurons. The number of3H-labeled ventricular zone cells above, rostral, or caudal to HVC was not affected by the hormone treatment MATERIAL AND METHODS in the short-survival birds, suggesting that testosterone did not affect neuronal production. However, the number of 3H- Animals. One-year-old female waterslager canaries from to robust of the our own colony were kept under conditions of the natural labeled HVC neurons that projected nucleus photoperiod in indoor facilities at the Rockefeller Universi- archistriatum (RA) in the long-survival birds was three times ty's Field Research Center. These birds were divided into greater in the hormone-treated than in the control group, two groups, the short-survival (n = 10) and the long-survival though the total number of RA-projecting cells did not change (n = 9) groups. The protocol followed with the short-survival signifcantly. Testosterone also induced an increase in the size birds was meant to quantify the possible effects of testoster- of the HVC cells that project to RA. Thus, these experiments one on VZ cell proliferation above, rostral, or caudal to HVC. suggest that testosterone affects the recruitment and/or sur- The protocol followed with the long-survival birds was meant vival of newly generated RA-projecting HVC neurons but does to quantify the effect of testosterone on the recruitment and not affect their production. survival of new HVC neurons. The short-survival birds were killed on October 9 and 10. The long-survival birds were Canaries learn their song by reference to auditory informa- killed on December 18 and 19. Because of the difference in tion (1). The high vocal center (HVC) is an important time ofyear when the birds were killed, our comparisons will component of the song system and is used in the acquisition be restricted to treatment groups that had the same survival. and production of learned song (2). Song is learned for the Treatment Protocols. Short-survival protocol. Birds in the first time during the months preceding sexual maturity and, short-survival group (Fig. 1A) received injections of the thereafter, modified every year (3). Most of these modifica- retrograde tracer Fluoro-Gold (F-G) (13) bilaterally into tions occur at the end ofsummer and in early fall (4, 5). These, nucleus robustus archistriatalis (RA) (14) on day 1. These too, are times of peak neuronal recruitment in the HVC of injections backfill only HVC neurons that project to RA, adult males (6, 7). Interestingly, the peak in neuronal recruit- which is part of the efferent pathway for the production of ment seen in late summer and early fall corresponds in adult learned song. RA-projecting cells backfilled with F-G defined males to a time when blood testosterone levels, which fell the boundaries of HVC. Each of these birds received on day during the summer, start to rise (5). 4 a subcutaneous Silastic implant (i.d., 0.76 mm; o.d., 1.65 Neurons added to the adult avian brain are born in the walls mm; length, 5 mm) that was either empty (controls, n = 5) or lining the lateral ventricle ofthe forebrain (8, 9), also referred filled with crystalline testosterone propionate (n = 5). Im- to as the "ventricular zone" (VZ). HVC is closely apposed plants of this size induce in adult female canaries blood to the ventrolateral wall of the lateral ventricle, and it has testosterone levels comparable to those seen in adult males been suggested that the neurons added to the adult HVC are in the reproductive condition (11). The implants were incu- born on the VZ overlying HVC (8). bated in physiological saline for 1 day before implantation to Female canaries sing rarely, and the song they produce has ensure immediate release of testosterone upon implantation fewer syllable types than that of males and is relatively (15). On day 6 of this protocol, each of the birds received a unstable (10). However, treatment of these birds with phys- single 50-14 i.m. injection of [3H]thymidine [6.7 Ci/mmol; iological doses oftestosterone induces them to sing more and New England Nuclear; z2.5 mCi/g (body weight); 1 Ci = 37 the song syllables they produce become louder and more GBq]. All birds in the short-survival group were killed on day 7 by an overdose of Nembutal followed by transcardiac The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: HVC, high vocal center; RA, nucleus robustus in accordance with 18 U.S.C. §1734 solely to indicate this fact. archistriatalis; VZ, ventricular zone; F-G, Fluoro-Gold. 7854 Downloaded by guest on September 26, 2021 Neurobiology: Rasika et A Proc. Natl. Acad. Sci. USA 91 (1994) 7855 was A Kill Background level determined with neuropil and was on average 0.019 grain per pm2. The minimum number of 3H- F-G RA T/O Siastic 3H-Thy labeled F-G-backfilled cells counted in a bird, both sides combined, was 42; the maximum was 245. Day I 4 6 7 The number ofcells per unit of HVC area was obtained by counting all cells of a given category (e.g., all F-G-backfilled cells or F-G cells labeled with [3H]thymidine) that appeared on a particular HVC section. These counts were obtained B Kill from four evenly spaced sections per side. In addition, 3H-Thy TIO Silastic Rep4ace F5G RA samples of 41-53 F-G-backfilled HVC cells were measured for each bird to obtain cell and nuclear diameters for 3H- Day 1-5 20 40 55 60 labeled and unlabeled cells. Nuclear size information was used to correct our counts and exclude the possibility that the FiG. 1. Schematic time table of experimental design. (A) Short- larger cells were sampled more frequently (19, 20). Counts survival protocol to test the effect oftestosterone on cell proliferation corrected in this manner were used to estimate total cell in the VZ. (B) Long-survival protocol to determine the effect of numbers. testosterone on the recruitment/survival of new neurons in HVC. When possible, we obtained data from both the right and T/O indicates testosterone-containing or empty Silastic implant. left hemispheres of the brain and compared the two sides. Since there was no systematic difference between the values perfusion with saline and then with 3% (wt/vol) paraformal- ofthe two sides, these birds were represented by the average dehyde. of the two sides. Where F-G injections on one side were Long-survival protocol. Birds in the long-survival group unsatisfactory, the bird was represented by the values ofonly (Fig. 1B) received one i.m. injection daily of 50 ILI of [3H]- the successfully backfilled hemisphere. thymidine on days 1-5. On day 20, they received a sub- All statistical comparisons between groups used the Mann- cutaneous Silastic implant that was either empty (n = 4) or Whitney U test. The paired Student t test was used for filled with testosterone (n = 5), as described above. These comparisons within groups. implants were replaced by another implant of the same kind on day 40 to maintain the levels of circulating testosterone. Choice of day 20 was determined by the observation that it RESULTS takes :20 days for HVC cells born in adulthood to become Short-Survival Birds. The VZ rostral to HVC had greater identifiable as mature neurons (16). Thus, testosterone treat- numbers of labeled cells per unit length of ventricular wall ment started when many of the cells produced during the 5 than the segments above HVC or caudal to HVC. However, days of [3H]thymidine injection were already differentiating the density oflabeled cells in any one ofthese regions was not into mature neurons and more were nearing the end of their significantly affected by the testosterone treatment (Fig. 2). migratory phase. Birds in the long-survival group received The mean number of silver grains per labeled VZ cell did not F-G injections into RA bilaterally on day 55 and were killed differ between testosterone-treated and control groups. on day 60 as described above. Testosterone treatment did not affect the volume of HVC Histolog. The brains ofbirds in both groups were removed as determined either in cresyl violet-stained (U = 10; P = after perfusion and placed in 3% paraformaldehyde for 3-4 0.602) or F-G-backfilled (U = 12; P = 0.917) sections. Cresyl days. Each brain hemisphere was embedded separately in violet and F-G volumes were very similar to each other (t = PEG (Mr, 1500) and sectioned sagittally at 6-jm intervals 2.045; P = 0.071). However, the nuclear diameters and soma (17). Every tenth section, used for autoradiography, was diameters of F-G-backfilled HVC neurons were larger in the mounted on chrome alum-coated slides, delipidized, coated testosterone-treated group than in the control group. Mean with NTB2 (Kodak) nuclear track emulsion, and incubated in soma diameter increased from 6.40 .um to 7.07 pHm (U = 0; P the dark at 40C for 4 weeks. These slides were then developed = 0.009); mean nuclear diameter increased from 5.97 pum to in D19 (Kodak) for 3 min at 170C and coverslipped with 6.43 jAm (U = 3; P = 0.047) (Fig. 3). Krystalon (Harleco, Philadelphia). Sections adjacent to each Long Survival Birds. Testosterone induced a significant section above were mounted on separate chrome alum- increase in the mean volume of HVC, as determined from coated slides, delipidized, and stained with cresyl violet. F-G F-G backfills (Figs. 4 and 5), which went from 0.09 mm3 in the was visualized in the sections used for autoradiography with control birds to 0.14 mm3 in the testosterone-treated birds, an UV fluorescence. increase of 60% (U = 2; P = 0.05). The volume increase in Data Analysis. All anatomical analysis was done using a the cresyl violet-stained material (67%) was comparable. computer-yoked microscope (18). The person doing the anal- There was no significant difference in the volume of HVC ysis did not know the treatment group to which each brain estimated by the two methods (t = 0.834; P = 0.428). belonged. HVC boundaries were mapped in the F-G and Soma diameters were larger in the F-G-backfilled HVC cresyl violet series and the area of HVC was calculated for cells of the testosterone-treated than in those of the control each section. The sum of these areas for each HVC, multi- birds. Cell diameters increased from 7.36 pm to 8.25 pAm (U plied by the section thickness and spacing between sections, = 0; P = 0.014). Nuclear diameters were also larger, but not allowed us to estimate the volume of HVC in short- and significantly (6.52-6.75 pum; U = 7.5; P = 0.54). When these long-survival birds. In short-survival birds, the strip of VZ comparisons are restricted to RA-projecting cells labeled seen in each section was divided into three segments- with [3H]thymidine, cell diameters increased from 7.72 pIm to directly above HVC, rostral to HVC, and caudal to HVC. 9.13 pim (U = 0; P = 0.014) and the nuclear diameters The length of each segment was measured, and [3H]thymi- increased from 6.76 jAm to 7.21 pum (U = 1.5; P = 0.04). It is dine-labeled VZ cells were counted. worth noting that the new (i.e., 3H-labeled) RA-projecting The position of all F-G-backfdlled HVC neurons-with or cells are systematically larger than the RA-projecting cells without 3H-labeling-was mapped in the long-survival birds not labeled with [3H]thymidine, regardless of treatment and the number of exposed silver grains over the nucleus of group (for soma diameters, t = -5.242 and P = 0.0008; for each ofthe labeled cells was recorded. The latter information nuclear diameters, t = -4.815 and P = 0.001). was obtained only for cells that had 30 times background level In addition, the packing density of F-G-backfilled cells in of labeling (minimum of seven grains per cell nucleus). HVC decreased from a mean of 119,000 cells per mm3 in the Downloaded by guest on September 26, 2021 7856 Neurobiology: Rasika et al. Proc. Natl. Acad. Sci. USA 91 (1994) A 0.14-

E 0.12- %-I 0.I-1- 0 E 0.08-

i 0.06- > 0.04- 0.02-

0- Backfill Cresyl FIG. 4. HVC volumes in testosterone-treated (hatched bars) and control (solid bars) birds of the long-survival experiment as deter- mined by F-G backfill or cresyl violet staining. Data are the mean ± B SEM. 12- DISCUSSION 11I Our observations on the short-survival birds showed that E testosterone treatment did not influence the number of cells E 8. I 1- dividing in the VZ above, rostral, or caudal to HVC. This 6 C result is similar to one reported earlier (12). However, we cannot conclude, from this alone, that testosterone does not -4 = affect the proliferation of HVC neuronal precursors because there is no direct evidence as to where these cells are. Moreover, testosterone might affect proliferation if given

-_ over longer periods of time. Testosterone did increase the Rostral Above HVC Caudal size of RA-projecting HVC neurons, an effect seen afterjust 3 days of treatment in the short-survival group, suggesting FIG. 2. Short-survival protocol. Effect of a testosterone- that testosterone reached the brain. In the long-survival containing or an empty Silastic implant on the proliferation of VZ cells. (A) Representative sagittal section indicating the regions of the group, the effect of testosterone on nuclear size was more VZ, rostral, above, and caudal to HVC, where 3H-labeled cells (black marked in the 3H-labeled than in the unlabeled neurons, dots) were counted and mapped. (B) Numbers of 3H-labeled cells in suggesting that testosterone affects HVC neurons differently these three VZ regions of testosterone-treated (hatched bars) and depending on their age, a fact that may lead to an under- control (solid bars) birds. Data are the mean ± SEM. standing of how testosterone acts to rescue dying neurons. We do not know when all the 3H-labeled RA-projecting birds to cells per mm3 in the testosterone- control 84,000 HVC neurons seen in the long-survival birds were born. treated ones. However, because of the differences in HVC Many of them resulted, presumably, from the division of the number of cells in HVC was volume, total RA-projecting precursor cells that were in S phase when [3H]thymidine was comparable between the two groups (10,600 vs. 11,500; U = administered. Some of those daughter cells could have di- 7; P = 0.46) (Fig. 6). vided again, and perhaps, testosterone encouraged them to Testosterone treatment induced a 3-fold increase in (Fig. 6) do so. However, had testosterone promoted the repeated the mean number of 3H-labeled HVC cells that to projected division ofneuronal precursor cells, then we would have seen RA-189 in the control group vs. 607 in the testosterone- relatively fewer numbers of exposed silver grains in the cell treated group (U = 1; P = 0.03). Labeled cells in both the nuclei of the testosterone-treated birds than in controls. Yet testosterone-treated and control groups were distributed the histogram ofthe number ofexposed silver grains over the throughout HVC. Fig. 7 shows that the number of exposed nuclei of HVC neurons backfllled with F-G was comparable silver over the nuclei of neurons was grains RA-projecting in the two treatment groups. Therefore, testosterone exerted comparable in the testosterone-treated and control birds. its effect by acting on postmitotic cells that did not divide again. P <0.05 Previous studies have shown that it takes newly formed HVC neurons -20 days to be born, migrate from their place of origin, and acquire a recognizable neuronal phenotype (9, >% 0.4- 0 16). Our testosterone treatment in the long-survival group c 0 started at that point, yet we saw 40 days later a 3-fold increase in the number of labeled RA-projecting neurons. From these

U- 0.2. results we infer that testosterone influences the recruitment Im and/or survival of new HVC neurons. An alternative expla- nation is that testosterone enhanced the transport of F-G toward the soma of 3H-labeled RA-projecting HVC neurons, 0- so that more of these cells were counted. There is no 3-4 4-5 5-6 6-7 7-8 8-9 9-10 for such an and it is that testoster- Nuclear diameter (pm) precedent effect unlikely one would have an effect exclusively in the cohort of cells FIG. 3. Distribution histograms of nuclear diameter of F-G- born during the period of [3H]thymidine treatment. If the backfilled HVC neurons in the testosterone-treated (hatched bars) effect was broader than this, then there should have been an and control (solid bars) birds of the short-survival experiment. The increase in the overall number of F-G-labeled HVC neurons, means (arrows) of these two distributions are significantly different. which was not the case. Downloaded by guest on September 26, 2021 Neurobiology: Rasika et al. Proc. Natl. Acad. Sci. USA 91 (1994) 7857

FIG. 5. (A and C) Fluorescent photomicrographs of HVC backfilled with F-G from RA in control (A) and testosterone-treated (C) birds. (B and D) Dark-field photomicrographs of the same fields in A and C, respectively, showing clusters of silver grains from 3H autoradiography. Note the increase in HVC size and in the number of 3H-labeled F-G neurons (arrows). (Bar = 100 ,um.) An earlier study reports that only one-third of the young whereas a majority of the neurons born in May disappear migratory neurons that move away from the VZ become fully during the next 4 months, there is no such reduction for differentiated adult neurons (9). Many ofthe newly generated neurons born in October (21). Testosterone or its metabolites cells presumably die before or soon after differentiation. could be responsible for this seasonal effect on survival. The Testosterone could increase the fraction of new HVC neu- level of testosterone in adult male canaries falls in the rons that survive during migration and that are drawn into summer and starts to rise in the early fall (5). The protocol we HVC-effects on recruitment. Testosterone may also in- followed in females may mimic this seasonal change in crease the survival of neurons after differentiation-an effect testosterone concentration seen in males. Whereas intact on survival. Our data cannot distinguish between effects on adult females have very low serum concentration of testos- recruitment or survival of HVC neurons. Testosterone may terone, a Silastic implant of the dimensions we used raises influence these processes directly or indirectly. Indirect serum testosterone levels to those seen in males in late fall (5, effects would occur if testosterone induced preexisting cells 11). The idea that testosterone promotes the survival of to produce factors that regulated the migration or survival of the new HVC neurons. The incorporation of new neurons into the HVC of adult 0.3 4-> . male canaries is higher in October than in May. In addition, C0.2~~~~~>

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0 T FIG. 7. Distribution histograms of number of silver grains for 3H-labeled F-G-backfilled HVC neurons in the testosterone-treated FIG. 6. Total number of F-G-backfilled HVC neurons and pro- (hatched bars) and control (open bars) birds of the long-survival portion of these cells that are 3H-labeled in testosterone-treated (bar experiment. The means (arrows) of these two distributions are not T) and control (bar 0) birds. Data are the mean ± SEM. significantly different. Downloaded by guest on September 26, 2021 7858 Neurobiology: Rasika et A Proc. Natl. Acad. Sci. USA 91 (1994)

newly formed HVC cells seems plausible because cells that 6. Alvarez-Buylla, A., Kim, J. R. & Nottebohm, F. (1990) Sci- concentrate testosterone or its metabolites occur in the HVC ence 249, 1444-1446. of adult female canaries (22, 23), and a subset of HVC's 7. Kim, J. R., O'Loulin, B., Kaspanian, S. & Nottebohm, F. androgen-concentrating cells project to RA (24). Still, it is (1994) Proc. NatIl. Acad. Sci. USA 91, 7844-7848. the present results were obtained from 8. Goldman, S. A. & Nottebohm, F. (1983) Proc. Natil. Acad. Sci. worth noting that USA 80, 2390-2394. intact adult females treated with [3H]thymidine and testos- 9. Alvarez-Buylla, A. & Nottebohm, F. (1988) Nature (London) terone in the fall. The outcome of our study might have been 335, 353-354. different if it had been conducted during other times of the 10. Weichel, K., Schwager, G., Heid, P., Guntinger, H. R. & year. Pesch, A. (1986) Ethology 73, 281-294. The incorporation of new neurons into brain circuits of 11. Nottebohm, F. (1980) Brain Res. 189, 429-436. juveniles and adults has been described in many vertebrates. 12. Brown, S. D., Johnson, F. & Bottier, S. W. (1993) J. Neurosci. The function of adult neurogenesis remains speculative; it 13, 2024-2032. may play a role in the modification of neural circuits (25-27). 13. Schmued, L. C. & Fallon, J. H. (1986) BrainRes. 377,147-154. In many, if not all animals, seasonal endocrine changes 14. Alvarez-Buylla, A., Theelen, M. & Nottebohm, F. (1988) Proc. accompany changes in life style, social status, stress, and Natil. Acad. Sci. USA 85, 8722-8726. reproductive state. These changes in behavior might require 15. Smith, E. R., Damassa, D. A. & Davidson, J. M. (1977) in ofthe underlying neural circuitry and Methods in Psychobiology, ed. Myers, R. D. (Academic, New dramatic modifications York), pp. 259-279. in some cases may involve the incorporation of new neurons. 16. Burd, G. D. & Nottebohm, F. (1985) J. Comp. Neurol. 240, Hormones are thus obvious candidates to regulate this form 143-152. of plasticity. For instance, recent evidence in mammals 17. Alvarez-Buylla, A. & Clayton, D. F. (1993) in Polyethylene indicates that adrenal hormones regulate cell division in the Glycol as an Embedmentfor Microscopy and Histochemistry, dentate gyrus of adult rats (28), a brain region where neuro- ed. Gao, K. (CRC, Boca Raton, FL), pp.71-80. genesis continues after birth (29, 30). 18. Alvarez-Buylla, A. & Vicario, D. S. (1988) J. Neurosci. Meth- The present work shows that agonadal-hormone influences ods 25, 165-173. the addition of neurons to an adult song control nucleus. 19. Weibel, E. R. (1979) Practical Methods for Biological Mor- Previous work in songbirds indicates that testosterone can phometry (Academy, New York), Vol. 1. also influence neuronal structure and synaptic number (31, 20. Clark, S. J., Cynx, J., Alvarez-Buylla, A., O'Loughlin, B. & 32). Thus, gonadal hormones may have a wide range of Nottebohm, F. (1990) J. Comp. Neurol. 301, 114-122. trophic effects on the neurons of juvenile and adult brains 21. Nottebohm, F., O'Loughlin, B., Gould, K., Yohay, K. & Alvarez-Buylla, A. (1994) Proc. Natl. Acad. Sci. USA 91, (33). The HVC ofadult canaries may serve as a model system 7849-7853. in which to study the molecular mechanisms by which 22. Brenowitz, E. A. & Arnold, A. P. (1990) J. Neurobiol. 21, testosterone exerts its trophic effects on the recruitment and 837-843. survival of neurons born in adulthood. 23. Gahr, M., Flugge, G. & Guttinger, H.-R. (1987) Brain Res. 402, 173-177. We thank Daun Jackson and Sharon Seppe for their expert care of 24. Johnson, F. & Bottjer, S. W. (1993) J. Neurobiol. 24, 400-418. birds and for helping with the [3H]thymidine injections. Arthur P. 25. Altman, J. (1967) The Neurosciences: First Study Program, Arnold, Carlos Lois, and M. E. Nottebohm made many helpful Neuroscience Research Program (Rockefeller Univ. Press, suggestions to our manuscript. This work was supported by National New York), pp. 723-743. Institutes of Health Grants MH18343 and NS28478. In addition, this 26. Nottebohm, F. (1985) in Hope for a New Neurology, ed. work benefited from the generous help of Mr. Herbert Singer, Mr. Nottebohm, F. (NY Acad. Sci., New York), pp. 143-161. Howard Phipps, and the Mary Flaggler Cary Charitable Trust. 27. Alvarez-Buylla, A. (1991) Exp. Neurol. 115, 110-114. 28. Gould, E., Cameron, H. A., Daniels, D. C., Wooley, C. S. & 1. Marler, P. & Waser, M. S. (1977) J. Comp. Physiol. Psychol. McEwen, B. S. (1992) J. Neurosci. 12, 3642-3650. 91, 8-16. 29. Bayer, S. A., Yackel, J. W. & Puri, P. S. (1982) Science 216, 2. Nottebohm, F., Stokes, T. M. & Leonard, C. M. (1976) J. 890-892. Comp. Neurol. 165, 457-486. 30. Kaplan, M. &-Bell, D. (1984) J. Neurosci. 4, 1429-1441. 3. Nottebohm, F. & Nottebohm, M. E. (1978) Z. 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