Brain Research 943 (2002) 80–92www.elsevier.com/ locate /bres

Research report

L esion induced new neuron incorporation in the adult hypothalamus ofthe avian brain

1 *Jie Cao , Karen Wenberg, Mei-Fang ChengBiopsychology Program, Department of Psychology, Rutgers University, 101 Warren St., Newark, NJ 07102, USA

Accepted 20 February 2002

Abstract

Cell loss in most adult vertebrate brain regions is thought to be irreversible. Here, we explore the effects of electrolytic lesions on theinduction of cell proliferation and newborn neurons in the ventromedial nuclei (VMN) of the hypothalamus in young and adult ring

3doves. The hypothalamus does not normally recruit new neurons. Bromodeoxyuridine (BrdU) and tritiated thymidine ([ H]Thy) wereused to identify cells born before and after bilateral electrolytic lesions. Hu and NeuN were used to identify neurons. TUNEL test forapoptosis and 3A7 antibodies were used to identify morphological changes of pre-existing cells. Lesions produced significantly morenewborn cells in the subventricular zone (SVZ). The rate of cell proliferation peaked at 7–14 days postlesion. A fraction of these newborncells were neuronal precursor and began to migrate away along the radial glial fibers 2 weeks after lesion. During this period, the outerarea of the lesion site was marked with massive apoptosis and re-expression of radial glial-like fibers. In birds that survived 5 months, wefound newly differentiated neurons in the outer area of the lesion site. We conclude that electrolytic lesion can invoke neuronalrecruitment in the adult hypothalamus. We further suggest that lesion-induced apoptosis and re-expression of developmental mechanismsmight be involved in the recruitment process. 2002 Elsevier Science B.V. All rights reserved.

Theme: Disorders of the nervous system

Topic: Trauma

Keywords: Adult neurogenesis; Hypothalamus

1 . Introduction transplantation of embryonic brain cells to the adult brainis associated with apoptosis induced developmental mecha-

Neuronal loss in the adult brain is widely thought to be nisms normally down regulated in the adult brainpermanent [1,8]. However, recent evidence indicates a [20,23,41,48]. This suggests that developmental mecha-more hopeful scenario. First, the adult brain retains the nisms are instrumental for successful neuronal replace-developmental potential to replace dying cells. For exam- ment. Self-repair in the adult brain, therefore, is within theple, wound-associated factors, such as transforming growth realm of possibility. Indeed, neuronal replacement wasfactor, epidermal growth factor, insulin like growth factor found in the high vocal center (HVC) in adult songbirdsand nerve growth factor, have been shown to facilitate cell following targeted neuronal death [40]. HVC is one of thedivision or cell survival of adult brain tissue in vitro telencephalic structures of the tetrapod vertebrates that[34,38,49] and in vivo [15]. Second, cell loss-induced normally incorporates neurons born in a distant region ofmicroenvironmental changes can activate developmental the adult brain [16,18,28,33,43]. Mechanisms for neuronalmechanisms in the adult brain. For example, successful recruitment are, therefore, already in place. Although the

finding of Scharff et al. [40] indicates that neuronalreplacement is not necessarily re-expressed in a stable cell

*Corresponding author. Tel.: 11-973-353-5851; fax: 11-973-353- population of HVC, Magavi et al. [29] reported evidence1171.

for in situ induction of neurogenesis in the neocortex ofE-mail address: mfc@psychology.rutgers.edu (M.-F. Cheng).1 adult mice where new neurons are produced at very lowPresent address: Monell Chemical Senses Center, 3500 Market St.,Philadelphia, PA 19104, USA. levels or not at all. These exciting findings leave open two

0006-8993/02/$ – see front matter 2002 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 02 )02537-4

J. Cao et al. / Brain Research 943 (2002) 80 –92 81

outstanding issues: (i) the functionality of adult neuro- 2 .2. Experimental proceduresgenesis is largely unknown; and (ii) the contribution of thesubventricular zone to adult neurogenesis has not been 2 .2.1. Short survival time study (Fig. 1A)established in regions that do not normally recruit new At 1 week before lesion, 3- and 8-month-old birds werecells. We set out to address some of these issues, using the injected with bromodeoxyuridine (BrdU) at 30 mg/kgadult hypothalamus that does not normally show neuro- bodyweight. At various intervals (3, 7, 14 or 30 days) after

3genic turnover. We have recently shown in the adult ring lesions, birds received a single injection of [ H]thymidine3dove that hypothalamic lesion-induced new neurons are ([ H]Thy, 4 mCi /kg body weight, 6.7 Ci /mmol). BrdU, an

3linked to recovery of co-specific electrical discharge in the analogue of thymidine [30], and [ H]Thy label replicatingregion [35]. Interestingly, recordings of normal physiologi- DNA, a necessary step before cell division [42,45] andcal response and appearance of new neurons occur 2–3 thus stain the nucleus of newborn cells. We used two cellmonths after lesions, the same time frame it normally takes division markers to distinguish cells born after the lesionsfor cells born in the subventricular zone to arrive in the from those born before lesions so that we could separateforebrain as differentiated neurons [27,33]. Here, we lesion induced cell division from spontaneously occurringstudied cell proliferative activity in the subventricular zone cell proliferation. The control birds received identicaland concurrent morphological changes at the lesion site, injections. Sham controls underwent the same surgicalfollowing bilateral lesion in the ventromedial nucleus procedure as those in lesion groups except that no electri-(VMN) of the hypothalamus of adult ring dove. The cal current was applied. Intact controls were not exposed tohypothalamus was chosen for this study for two reasons: surgical procedures. All birds were perfused 24 h after the(i) this region is an area that does not normally incorporate second injection.new neurons in the adult brain [26,32]; and (ii) extensiveknowledge of specific behavioral and physiological prop- 2 .2.2. Long survival time study (Fig. 1B)erties of the hypothalamus of the ring dove [11,12] enables In this study, 3- and 8-month-old birds received identi-us to relate the present finding in a meaningful context. We cal injections to the short survival study, except that all

3used well-characterized Hu antibodies and markers for birds received the second injection (single [ H]Thy) 1newly generated cells to identify and monitor neuronal week after lesions and were allowed to survive for 5precursor cells before and after lesions in the subventricu- months before perfusion.lar zone (short-term survival study). Morphological Hu proteins, a neuron-specific family of RNA bindingchanges at the lesion site were monitored by TUNEL test proteins in vertebrates [44], are also expressed in prolifer-and 3A7 antibodies for apoptosis and astroglial cells. ating neurogenic cells [6,30]. We thus used Hu antibodiesNeurons were identified by Hu and NeuN antibodies (long- as the marker for neuronal precursor cells in the shortterm survival study). We found lesion-induced cell prolifer- survival study when cells are to be found in the vicinity ofation in the subventricular zone, cell migration and succes- the subventricular zone where they are born, and as asive morphological cell changes at the lesion site that marker for mature neurons in the long survival studyculminate in neuronal incorporation in the adult hypo- where surviving cells with neuronal properties can bethalamus. detected. In the long survival time study, in addition to the

Hu antibodies, selective samples of lesioned and controlgroups were also subject to anti-NeuN, a transcriptionfactor that is expressed in the nucleus and cytoplasm of

2 . Materials and methods mature neurons [32].

2 .1. Animals 2 .2.3. Cell migration study (Fig. 1C)To monitor the migration of newborn cells, 3-month-old

Male 3- (young) and 8-month-old (adult) ring doves birds were injected with BrdU at 1, 3 or 7 days postlesion(Streptopelia risoria) were subjects in all except the and were allowed to survive 3, 7, 14, or 30 days postle-migration experiments (the numbers of subjects in all sion. Each survival group was subjected to 3A7 immuno-groups, except the cell death study, are listed in Table 1). staining to identify radial glial cells. We measured the

1Females were not included in this initial study, because a shortest distance between the position of BrdU cells andfemale’s variable reproductive phase may complicate the the ventricle, using image analysis software ImagePro.experimental design. At 3 months old, ring doves are 3A7 is an IgM type monoclonal antibody that specificallycomparable to adult birds in body weight but have gonads binds to intermediate filaments of the embryonic chickenthat are just beginning rapid growth. At 7 months old, ring glial cells [21,24]; in adult birds, 3A7 preferentially labelsdoves are reproductively mature. All birds were bred in the radial glia [17].indoor colony at the USDA certified Rutgers AnimalFacilities and maintained with constant room temperature 2 .2.4. Cell death studyof 22 8C and 12:12-h light–dark cycle. The number of dying cells (apoptotic cells) following

82 J. Cao et al. / Brain Research 943 (2002) 80 –92

Table 1Mean number of cells in both studies

aGroup Cell type Subjects1 3 1 3 1 1BrdU [ H]Thy [ H]Thy /Hu

(mean6S.D.) (mean6S.D.) (mean6S.D.)

25 b(a) Mean number of cells (in cells 310 /mm) identified by different cell type markers in short survival study (telencephalic SVZ)A 3mon3d(1) 214.6611.9 329.4617.8 53.367.2 3B 3mon7d(1) 333.6624.6 455.9614.2 81.267.6 6C 3mon14d(1) 276.8616.4 433.4621.6 78.068.1 6D 3mon30d(1) 252.9617.8 310.9627.5 48.266.4 3E 3mon10dSham(2) 161.5614.8 236.8616.7 34.961.7 6F 3mon7dIntact(2) 167.8613.9 259.0616.0 37.666.3 3G 8mon7d(1) 187.7621.3 266.0627.9 43.1611.4 8H 8mon7dSham(2) 146.3620.5 183.5618.7 27.366.5 3I 8mon7dIntact(2) 140.1623.2 197.7621.6 29.267.3 2

25(b) Mean number of cells (in cells 310 /mm) identified by different cell type markers in long survival study (telencephalon and hypothalamusparenchyma)J 3mon3d(1) 77.267.0 152.4622.2 17.663.1 5K 3mon7d(1) 68.1610.7 177.9654.9 15.363.4 4L 3mon14d(1) 69.8614.4 108.6628.5 23.767.3 4M 3mon30d(1) 67.068.2 98.1621.9 15.563.5 6N 3mon10dSham(2) 72.768.6 73.467.1 15.864.5 3O 3mon14dIntact(2) 68.961.7 104.2619.0 16.161.6 3P 3mon7dIntact(2) 62.461.9 86.066.3 15.061.2 3Q 8mon3d(1) 52.866.3 64.163.3 14.662.3 4R 8mon7d(1) 70.2611.8 92.7611.8 22.663.8 7S 8mon10dSham(2) 63.368.1 73.966.5 16.762.7 6T 8mon7dIntact(2) 53.267.6 84.3610.5 17.966.0 4a 3The first numerical number in each group refers to the age of the birds, the second number refers to the number of days between lesion and [ H]thymidineinjection, the 1 /2sign refers to lesion. In the case of the control group (Sham and Intact), the second number refers to the number of days between BrdU

3 1 3 1 3 1 1and [ H]thymidine injections. BrdU refers to cells born before lesion; [ H]Thy refers to cells born after lesion; [ H]Thy /Hu refers to double-labelednewly generated neuronal precursor cells after lesion; in long term survival groups, these double labeled cells represent differentiated new neurons.

1 3 1 3 1 1ANOVA for BrdU , [ H]Thy , and [ H]Thy /Hu , respectively, are: F5151,63, df 18,65, P,0.0001; F5143,97, df 18,65, P,0.0001; F556.40, df18,65, P,0.0001.b Examination of the SVZ of the third ventricle detected very sparse new cells with or without lesions, hence cell count was made only for the telencephalicSVZ.

lesions was monitored using the TUNEL reaction. Both 3- ance with a protocol approved by Rutger’s Animal Careand 8-month-old birds were perfused at 3, 7, 14 or 30 days and Facilities Committee.postlesion.

2 .4. Immunocytochemistry and autoradiography

2 .3. Electrolytic lesions 2 .4.1. Perfusion and paraffin embeddingBirds were perfused with 100 ml 0.9% saline, then 200

Bilateral electrolytic lesions in the ventromedial nuclei ml cold 4% paraformaldehyde (PFA) in 0.1 M phosphate(VMN) of the hypothalamus were performed on ex- buffer (PB) (pH 7.4). Their brains were removed andperimental birds under anesthesia (Chloropent; 2.5 ml /kg). embedded in the same fixative overnight at 4 8C. On theThe head of the bird was fixed on a stereotaxic instrument 2nd day, the brains were dehydrated through 50% EtOH (2at an angle of 458. After the skull was exposed, a small h), then further dehydrated through 70%, 95%, and twohole (diameter |3 mm) was drilled in the area corre- times each of 100% EtOH (2 h); they were then delipid-sponding to the coordinates for the VMN area (AP, 2.6; L, ized with xylene (4 h) and finally placed into paraffin that0.5; D, 8.5 mm) [22]. An insulated 0.2-mm O.D. tungsten was pre-warmed to 50–55 8C. Brains were kept in eachwire electrode with only the tip exposed was lowered to solution with several changes, then transferred to a plasticthe VMN area. A 1-mA constant positive current was mold oriented to the cutting angle, and then kept in aapplied for 40 s to produce a lesion of |1.5 mm in refrigerator.diameter (Fig. 2A). The wound was sutured, and the birdwas returned to its original cage. 2 .4.2. Sectioning and mounting

All the intrusive procedures were conducted in accord- (i) Brains in the short- and long-term survival studies

J. Cao et al. / Brain Research 943 (2002) 80 –92 83

Fig. 1. Experimental flow charts. (A) Short survival time. (B) Longsurvival time. (C) Cell migration study.

were fixed in paraffin and were cut (coronal sections) on anHM350 (Microm) rotary microtome at 6-mm thickness.

Fig. 2. Photomicrographs showing the brain lesion site and different cellFor each immunostaining, every 50th section wastypes in the subventricular zone of an adult male ring dove. (A) Bilateralmounted, but alternate consecutive sections were used forlesions in the VMN, necrosis on the left and cavity on the right. (B)

different markers. Sections were mounted from warm Different markers for identifying different types of cells along the SVZ 71phosphate buffered saline (PBS) onto gelatin-coated days postlesion. Cells born before lesion (BrdU cells, dark brown,

3 1microscope slides, then heat adhered overnight at 40 8C. indicated by filled arrowheads), cells born after lesion ([ H]Thy cells,1dark grains indicated by filled arrows), neuronal precursor cells (Hu(ii) Brains in the cell migration study were fixed in 4%

cells, light brown, indicated by hollow arrowheads), and neuronalPFA–0.1 M PB (pH 7.4) overnight at 4 8C, then in 20% 3 1 1precursor cells born after lesion ([ H]Thy /Hu cells, dark grains oversucrose–PBS solution overnight at 4 8C. Brains were light brown indicated by a filled arrow and a hollow arrowhead). Scalesectioned at 10-mm thickness, using a cryostat microtome. bar represents 500 mm (A) and 20 mm (B). LV, the lateral ventricle; OC,The sections were thaw-mounted onto slides and stored in optic chiasm; V, the third ventricle.

a 280 8C freezer until use.

2 .4.3. Immunostaining primary antibody (mouse monoclonal antibodies, 1:200The paraffin slides were first deparaffinized through a anti-BrdU; Boehringer Mannheim). PBS containing 2%

series of xylene (three changes), 100% (two changes), normal horse serum and 0.2% Triton X-100 was added at95%, 80%, 70% EtOH and finally PBS, each for 15 min. 100 ml /slide, covered with parafilm, and incubated over-For BrdU detection, the sections were first hydrolyzed by 2 night at 4 8C. Slides were then washed in PBS 335 minN HCl for 30 min at 37 8C, washed twice with borate and processed according to the instructions with Vector’sbuffer (0.1 M, pH 8.5) for 15 min, and washed with PBS ABC kit. Color development reactions were carried out in(335 min) at room temperature before anti-BrdU mono- PBS solution containing 0.025% DAB, 0.04% NiCl ,2

clonal antibody was added. For immunocytochemistry, the 0.01% H O for 5–10 min at room temperature.2 2

slides were washed in PBS then incubated in the first For double labeling, the slides were washed in PBS for 5

84 J. Cao et al. / Brain Research 943 (2002) 80 –92

times. Then the second primary antibody (1:500 mouse slide. The sections were mounted and the slides coveredwith glass coverslips.monoclonal antibody (clone 16A11), anti-Hu (a gift from

Drs S.A. Goldman and H. Furneaux) for the short survival2 .6. Cell counting and data analysisstudy, or 1:100 NeuN (a gift from Dr R.J. Mullen) for the

long survival study) was added and the same procedureCell counting and data collection were done with afollowed as above, except that the DAB concentration was

computer aided microscopic system [2]. For the birds of0.05%, and NiCl was not added. The immunohistochem-2

the short survival study (birds killed 1 day after singleistry for 3A7 staining was the same except that monoclonal3[ H]Thy injection), the entire SVZ region was counted forIgM antibody 3A7 [24] was used at 1:20 in 2% normal3 1 3 1 1horse serum PBS solution; then the same procedure for [ H]Thy and [ H]Thy /Hu double-labeled cells. The

BrdU immunostaining was used. When the immunostain- length of the lateral ventricle was measured in each case.ing reaction was completed, the slides were dehydrated The cell density is the number of specific type of cells perthrough a series of increasing concentrations of alcohol unit length of the lateral ventricle. Three representativefollowed by two changes of xylene, and then coverslipped sections of |300–600-mm intervals were selected forfor observation. counting. From rostral to caudal, the first plane, plane A,

was chosen where the highest cell proliferation were seen(the so-called ‘hot spot’ area [4,27]) which corresponds to

2 .5. Autoradiographytransverse level of A10.0 of the pigeon brain atlas [22].The second plane (plane B), was at the level of the anterior3After immunocytochemistry, to detect [ H]Thy labeledcommissure (A7.75). The third (plane C) was at the level

cells, the slides were dipped in KODAK NTB-2 photoof the lesion site, the VMN (A2.6). For birds in the long

emulsion pre-warmed to 42 8C in the dark, air dried for 3survival study (killed 5 months after single [ H]Thy2–3 h, then collected into light-tight black boxes with

injection), the entire telencephalon was counted for each ofdesiccant in them and exposed for 3 weeks in a cold room,

the three planes. For all birds, three types of cells wereThe slides were developed with KODAK D-19 for 3 min at 1counted: the BrdU labeled cells (BrdU cells, dark brown17 8C, stopped by KODAK liquid hardener (Cat. [146 3 1 3color), [ H]Thy labeled cells ([ H]Thy cells, black dots4239), fixed in KODAK fixer (Cat. [197 1746) for 15 min 3 1 1of silver grain) and [ H]Thy /Hu double-labeled cellsat 20 8C, rinsed with plenty of water, and then dehydrated

(black dots of silver grain with light brown cell body). Aby going through increasing EtOH series and coverslipped

similar counting method was used in the cell deathwith permount (Fisher Scientific, SP15-500).

experiment.The specificity of each of the antibodies used in this 3The criterion for [ H]thymidine labeled cells is the same

study has been validated in the adult birds: Hu antibodiesas those accepted in the literature, that is, the existence of

[6], NeuN [27], BrdU [31], and 3A7 [17]. A control slideseven or more exposed silver grains over the nucleus,

(to which the primary antibody was not added) waswhich is ten or more times above background [3]. In the

included in each experiment for control of specificity.present study, the labeled cells we included in countingcontained more than 20 silver grains.

2 .5.1. TUNEL reaction For brain parenchyma, including the telencephalon andFrozen sections were cut on the cryostat microtome at the hypothalamus, the mean cell density of the three

10-mm thickness, and thaw-mounted onto gelatin coated counting planes combined was used as the data point forslides without cover. The slides were air-dried, then all comparisons. For the hypothalamus, cell counts werewashed in PBS for 5 min at room temperature. They were made within a 2-mm (height)31-mm (width) area of thethen covered with 100 ml equilibration buffer (200 mM ventral medial hypothalamus. For telencephalon SVZ cellpotassium cacodylate, pH 6.6, at 25 8C), 25 mM Tris–HCl counts, the unbiased cell number of each section was first(pH 6.6 at 25 8C), 0.2 mM DTT, 0.25 mg/ml BSA, 2.5 normalized to cell density (numbers of cells per unit lengthmM cobalt chloride, and equilibrated at room temperature of the lateral ventricle), then the mean of the cell density of

2for 5–10 min. For each 5-cm area, 50 ml of TdT the three planes combined was determined for each bird. Inincubation buffer (45 ml equilibration buffer, 5 ml Nucleo- all cell counts the unbiased estimate of the total celltide mix (50 mM fluorescein-12-dUTP, 100 mM dATP, 1 number was made using the stereological /physical dissec-mM EDTA, 10 mM Tris–HCl, pH 7.6), 1 ml TdT enzyme, tor method [13].from Promega) were added. The slides were covered with All cell counts and measurements were recorded withplastic coverslips and incubated at 37 8C for 60 min in a MS Excel program. The statistical analysis was carried outhumidified dark chamber. Reactions were terminated by with StatView, a statistical analysis program from SASimmersing the slides in 23SSC for 15 min at room Institute, on a Macintosh computer. ANOVA and post-hoctemperature, removing coverslips, and then were washed in Fisher’s protected least significant difference test werePBS for 335 min. One drop of Anti-Fade solution used to analyze the differences among the experimental(Molecular Probes, Cat. [ S7461) was added to each conditions.

J. Cao et al. / Brain Research 943 (2002) 80 –92 85

3 . Results lesions, yielded a significantly greater number of newlygenerated neuronal precursor cells than adult brains (Fig.

3 1 1Table 1a and b summarizes cell counts of various cell 3, 3-month-old vs. 8-month-old birds, [ H]Thy /Hu :1classes that are the basis for statistical analysis. For clarity, P,0.05). Interestingly, more BrdU cells (born before

data are presented in two sections: (a) short-term survival lesion) were seen in the lesioned group than in the controland (b) long-term survival. group (Fig. 3, P,0.05 for both age groups), indicating that

the lesion also enhanced proliferative activity or survival3 .1. Effects of lesion on cell proliferation and the rate of cells born before the lesion.of cell death in SVZ Fig. 4A shows the distribution of neuronal precursor

cells in the SVZ 7 days after lesions. Most new cells wereFollowing bilateral lesions in the VMN (Fig. 2A), cells born at the anterior tip of the SVZ, the ‘hot spot’ as in the

born before lesions and cells born 7 days after lesions, can normal adult ring dove brain [27]. We did not detect any3 1 1be seen along the SVZ (Fig. 2B). Examination of the focal [ H]Thy /Hu cells along the third ventricle in either

SVZ (three planes used in counting; see Methods section) lesioned or control birds. There were no double-labeledof both the young and adult birds, shows a significantly cells in the telencephalon 7 days after cells proliferated ingreater number of newborn cells in lesioned brains, the SVZ. However, double-labeled cells can be seen

3 1compared to that of control brains (Fig. 3, [ H]Thy cells scattered in the telencephalon in 5-month postlesion brainin lesioned vs. control: P,0.05 for both age groups). What (Fig. 4B).fraction of the newborn cells were neuronal precursorcells? For 3-month-old birds, the percentage of newly 3 .2. Time course of lesion-induced newborn cells in SVZ

3 1 1generated neuronal precursor cells ([ H]Thy /Hu )3 1ranged from 16 to 18% for the lesioned (Table 1a, groups Lesion-induced newborn cells ([ H]Thy ) were evident

A–D) and 14% for the control (groups E, F) groups, while on day 3 after lesions, but the number peaked between 7the corresponding percentages for 8-month-old birds were and 14 days postlesion and tapered off thereafter (Table16 and 14% (Table 1a, group G vs. H, I). Young birds 1a, groups A–D). At 30 days after lesions, the SVZ of thealready generating a greater number of new neurons, after lesioned brain was still producing more new cells than that

Fig. 3. Comparison between the lesioned and control groups of young and adult birds: the number of cells born in the SVZ before and 7 days postlesion.Data of groups compared in this histogram are listed in Table 1a. Comparisons between the lesioned and control groups were made at comparable timepoints. Lesioned groups consist of group B (3-month-old birds) and group G (8-month-old birds), both 7 days postlesion. The 3-month-old control groupconsists of group E (sham, 10 days postlesion) and group F (Intact, 7 days). The 8-month-old control group consists of group H (Sham, 7 days) and group I(Intact, 7 days). Sham and Intact groups were combined as control groups as they were not statistically different from each other. See text for Fisher’s

1 3 1 3 1 1PLSD test result. BrdU cells: new cells born before lesion; [ H]Thy cells: new cells born after lesion; [ H]Thy /Hu cells: newly generated neuronalprecursor cells, born after lesion.

86 J. Cao et al. / Brain Research 943 (2002) 80 –92

Fig. 5. Histogram and estimated distribution curve showing migrationdistance of newborn cells of 3-month-old birds at 1 week postlesion in thesubventricular zone (SVZ) (A) and 1 month postlesion in the brainparenchyma (B) (see Methods section for measurement). Data are basedon 16 birds, four in each of four survival times.

however, most newborn cells were located 200–300 mmaway from the ventricle (Fig. 5).

3 .4. Effects of lesion on the number of newborn neuronsin the telencephalon

At 5 months after bilateral lesions of the VMN, the3 1 1number of newborn neurons ([ H]Thy /Hu ) found in theFig. 4. A representative map showing the distribution of the newly

3 1 1 whole telencephalon (represented by three focal planes)generated cells. Neuronal precursor cells ([ H]Thy /Hu ) 1 week3 1 1 was more than that of the control brains (Table 1b,postlesion (A) and newborn neurons ([ H]Thy /Hu ) 5 months postle-

sion (B). B is based on bird [4142, 8 months old and surviving 5 months 3-month-old birds, group L vs. O and 8-month-old group R3after lesion and a single injection of [ H]Thy. Clusters of black dots vs. S: P,0.05 for both age groups). To determine the

denote the areas where newborn neuronal precursor cells (A) or newbornnumber of cells born in the SVZ that subsequentlyneurons (B) were found in five out of ten sections of 6-mm thickness. CA,differentiated into neurons, we compared the number ofanterior commissure; E, ectostriatum; HV, hyperstriatum ventral; LPO,

3 1cells born ([ H]Thy ) in the SVZ 3 days after lesionlobus parolfactory; N, neostriatum; Rt, nucleus rotunda; V, lateral3 1 1ventricle. versus newborn neurons ([ H]Thy /Hu ) found in the

brain telencephalon 5 months later. The comparison isjustified since both these experiments (short-term and long-

of the control brain of the 3-month-old birds (Table 1a, term survival paradigms) involve one single injection ofgroup D vs. E: P,0.01). For the 8-month-old birds, there tritiated thymidine. In control brains of 3-month-old birds,were only two postlesion data points, however, the result only 15% of new cells born in SVZ survived as mature

1 1shows the same trend (Table 1a, group G vs. H). neurons (Hu or NeuN ) 5 months later (Table 1b, group3 1 3 1 1O, [ H]Thy vs. [ H]Thy /Hu ). The percentage for the

3 .3. Time course of cell migration lesioned brains was higher at 22% (group L). In controlbrains of 8-month-old birds, 23% of the cells born in SVZ

3Cell migration was monitored in 3-month-old birds. We survived as mature neurons 5 months after [ H]Thyestimated the location of each newborn cell by measuring injection (group S); the corresponding number for theits shortest distance from the surface of the SVZ. The brain with lesions was 24% (group R). Interestingly, evendistribution pattern of BrdU labeled cells at different though the 3-month-old birds with lesions yielded sig-intervals from the time of lesioning shows a clear shift in nificantly more SVZ newborn cells (birds killed 24 h after

3location away from the SVZ (note: in this experiment [ H]Thy injection) than the 8-month-old birds with lesions,BrdU was used to label newborn cells after lesions.) the number of surviving neurons 5 months after lesionsDuring the 1st week following lesion, the newborn cells was not different between the two age groups (Table 1b,were congregating in or near the SVZ. By 2 weeks, group L vs. R: 2317.3 vs. 22.613.8, P.0.05).

J. Cao et al. / Brain Research 943 (2002) 80 –92 87

3 .5. Cell death, re-expression of developmentalmorphology, appearance of newborn cells and neuronsin the hypothalamus

Massive apoptotic cell death was seen in and around thelesion site 24 h (the earliest data point we collected)following the VMN lesions. Cell death in this regionsoared to 80-fold compared to that of the control brain, butdeclined precipitously by 2 weeks postlesion (Fig. 6). Incontrast, lesions did not appear to have an impact on therate of programmed cell death in the SVZ: the low numberof TUNEL-labeled cells in SVZ remained unchanged afterlesions (Fig. 6).

Interfacing with the tapestry of apoptotic cells wasremarkable re-expression of the developmental morpholo-

1gy. At 1 day postlesion, astrocyte like cells (3A7 )populated the outskirts of the lesion site, a |400–500-mmzone extending from its rim (Fig. 7B). By the end of 1month postlesion, the astrocyte-like cells in the region

1were displaced by long non-branching 3A7 fibers (Fig.7D), similar to radial glial fibers seen along the SVZ (Fig.7A,C). These morphological changes were not seen incontrol brains (Fig. 8). Parallel to the dramatic microen-

Fig. 7. Photomicrographs of coronal sections showing changes in andaround the SVZ (A, C, E) and the hypothalamus (B, D, F) after lesions of8-month-old birds. At 1 day postlesion, radial glial cells with long fibers

1(3A7 , indicated by arrows) were present along SVZ (A: v, ventricle),1whereas stellated cells (3A7 , indicated by arrowheads) populated the

1outer boundary of the VMN (B: arrowhead represents NeuN cell; dashedline represents the brim of the lesion site). At 2 weeks postlesion, theneuronal precursor cells began to migrate guided by radial glial fibersalong SVZ (C), and the outer boundary of VMN was populated by radial

1glial like fiber (3A7 , indicated by arrows) (D: dashed lines represent thebrim of the lesion site). At 1 month postlesion, newborn cells (indicatedby arrowhead) were seen coasting along radial glial fibers (indicated byarrows) |500 mm away from the SVZ (E). At 5 month postlesion,

3 1 1newborn neurons ([ H]Thy /NeuN , indicated by an arrow; arrowhead1represents NeuN ) were detected 500 mm lateral to the lesion site (F).

Scale bars represent 100 mm (A, B, C, D), and 20 mm (E, F).

vironmental changes in the outskirts of the lesion site wasthe timely appearance of newborn cells and newborn

3 1neurons in this region. The expression of [ H]Thy cells(most likely the lesion-induced gliosis) was detected at day3 postlesion (the earliest data point on thymidine labeling).

1 1Newborn cells along radial glial-like fibers (BrdU /3A7 )were detected in the outskirts of lesion site 1 month

3 1 1postlesion (Fig. 7E). Newborn neurons ([ H]Thy /Hu )were detected only in long-term survival groups of youngand adult birds (Fig. 7F); they were not expressed in brainsat days 3, 7, 14 or 30 days postlesion. In contrast, thehypothalamus of the control brain of both young and adult

3 1 3 1 1birds was devoid of [ H]Thy or [ H]Thy /Hu cells.Fig. 6. Time course of lesion induced apoptotic cells in different brain Fig. 9 summarizes morphological changes taking placeregions of adult birds. Each data point is based on n54 (two subjects, two

postlesion within 400–500-mm outskirts of the lesion site.sections each). ‘0’ represents intact birds serving as control birds. In 3 1To assess the number of newborn neurons ([ H]Thy /defining the borders of the regions, we adopted those set forth by1

Devoogd et al. [14]. Hu ) being recruited into the hypothalamus, we used the

88 J. Cao et al. / Brain Research 943 (2002) 80 –92

Fig. 9. Photomicrographs showing the distribution of the radial glial likefibers, the apoptotic cells and new neurons at the outer area of a lesion

1site. 3A7 fibers (indicated by black arrows) radiated from the lesion site;1hollow arrowheads indicate newborn cells (BrdU ) (A). Apoptotic cells

(white arrowheads), identified by fluorescent TUNEL method, alsopopulated the same region (B). One of the new neurons labeled by

3 1 1[ H]Thy /Hu in the region (C) is magnified in (D). Solid linesrepresent the brim of the lesion site, dashed lines represent the outerboundary of focal area which measures 400–500 mm from the center ofthe lesions. Scale bar represents 20 mm (D) and 100 mm (A–C).

that in the ectostriatum (346.2629.7 vs. 92.4616.0 cells /3mm ).

Fig. 8. Photomicrographs showing various control groups. (A–D) Con-trol groups for Fig. 7. (A) No labeling for 3A7 labeling in VMN of an 4 . Discussionintact bird (bird [4343); (B) no labeling for 3A7 labeling in VMN 1week after sham lesion (bird [4341); (C) no labeling for 3A7 labeling in

The current study was undertaken to assess the effects ofVMN 1 month after sham lesion (bird [4335); (D) absence of3 1 1[ H]Thy /NeuN double labeling in VMN 5 months after sham lesion electrolytic lesions on cellular changes including neuro-

(bird [4142). (E–H) Control slides for various immunostainings where genesis in the adult brain. Specifically, we examinedprimary antibody was omitted. (E) BrdU (bird [4240); (F) Hu (bird

whether an adult hypothalamus that normally does not add[3998); (G) NeuN (bird [4240); (H) 3A7 (bird [4526). Scale barsnew neurons [16,27,33] would, as a result of lesion-represent 100 mm (A–C, E–G) and 20 mm (D).induced cell loss and related events, undergo changesfavorable to recruiting cells born in the SVZ. We adoptedthe short survival paradigm (birds killed within days after

3unbiased stereological method [13] to count the total [ H]Thy administration) for monitoring proliferative activi-number of newborn neurons in ten sections of 6-mm ty in the SVZ, and the long survival paradigm (birds killed

3thickness (300 mm apart) in the VMN of each brain. The 5 months after [ H]Thy injection) for detection of newbornVMN area counted was 2 mm high31 mm wide (1 mm neurons in the parenchyma. In both paradigms, we injectedfrom the third ventricle midline and 2 mm from the optic only one single dose of tritiated thymidine. The singlechiasm). For reference comparison, we counted cells in an injection in the long survival study is a significant de-area 2 mm high31 mm wide in the center of the nucleus parture from other published work using this methodologyectostriatum, a well-circumscribed visual nucleus in the [3,4,6,16,25,27,43,46]. Multiple injections over a numbertelencephalon. Fig. 10 summarizes the results of these cell of days and weeks are the rule. Although far fewercounts (in volume) of the 8-month-old birds. The mean newborn cells are labeled than with a single injection, the

3 1 1 3number of [ H]Thy /Hu cells was 1714.9 cells /mm in paradigm has the advantage of allowing us to compare5-month postlesion brains. There was a slight but negli- short and long survival groups. Labeled cells in the presentgible increase of newborn neurons in the ectostriatum study are therefore a very conservative estimate of lesion-(P.0.05). A sharply higher number of newly proliferated induced newborn cells. In ring doves, most of the labeling

3cells were also found in the hypothalamus compared to from a single injection of [ H]Thy occurs during the first

J. Cao et al. / Brain Research 943 (2002) 80 –92 89

astrocytes to the radial glial cells and eventual incorpora-tion of the new neurons, occur within a 400–500-mmboundary from the center of the lesion. This is the samearea where we previously recorded normal neuronal firingsin adult brains 2–3 months following lesions [35]. Theelevation of apoptotic cells after lesions is significant intwo respects: (i) the data confirm an earlier finding on theadult gymnotiform brain that although CNS cells undergonecrosis immediately following a lesion, a few hourspostlesioning apoptosis is the predominant type of celldeath [50]; and (ii) apoptotic cells are thought to triggerneuronal replacement in the forebrain of the adult songbird[40] and in the neocortex of adult mice [29]. Thus, fromthe elevation of apoptotic cells in the hypothalamusfollowing lesions, it may be construed that signals fromthese cells may be involved in the induction of lesion-induced cell proliferation.The morphological changes of

13A7 cells in the outer boundary of the lesion site areequally remarkable: cells were stellated 1 day postlesion(Fig. 7B) and were elongated with non-branching fibersradiating from the brim of the lesion site 1 month later(Fig. 9A). Based on the findings of Leavitt et al. [23] thatmature astrocytes can transform into transitional radial gliato support directed migration of transplanted immatureneurons [23], we suggest that the morphological cellchanges seen in the outer boundary of the lesion site mayreflect a similar mechanism.

Fig. 10. A histogram showing the mean number of newborn neurons An increase in the number of newly proliferated cells3 1 1([ H]Thy /Hu cells) in the hypothalamus and the ectostriatum 5 months was detected in the SVZ 3 days following lesions, of3 1 1postlesion of adult birds. [ H]Thy /Hu cell counts were done within the

which only a fraction were subsequently double labeledrectangle counting area shown on the map. N, neostriatum; OC, opticwith a neuron specific marker. By 2 weeks after thischiasm; V, ventricle; VMN, ventromedial nucleus (also called PMH).N3 1 1 3 ]][ H]Thy /Hu cell density in the counting area (cell /mm ) 5 increase, some cells were seen along guiding radial glialA 3 T3 1 1where N is unbiased estimate of the total [ H]Thy /NeuN cells in the fibers indicating that these cells were migrating. The time

counting area by physical dissector method [13], A is counting area which course of cell migration in 3-month-old birds suggests thatis 2 mm31 mm (for both the hypothalamus and the ectostriatum) and T

most newborn cells were located 200–300 mm from theis section thickness which is 6 mm.ventricle 1 month after lesions. At this rate, it would take|2 months for these cells to reach the hypothalamus.Indeed, we were unable to detect any newborn neurons in

half hour [26]. This means that the newborn neurons the young or adult hypothalamus 1.5 months postlesion. Inobserved 5 months after a single injection represent only a the study on adult mice by Magavi et al. [29], it was30-min snapshot of 2-week-long activity of highly elevated suggested that endogenous neuronal precursors were in-cell proliferation after the lesions. Newborn neurons in the duced to differentiate in situ without ruling out the possiblehypothalamus were few in number, as we had expected, contribution from the SVZ. On the other hand, in the studybut the number of cells was respectable compared to a on adult songbirds by Scharff et al. [40], it was shown thattelencephalic structure, the ectostriatum, which normally neuronal replacement following targeted lesions in the highrecruits new neurons (Fig. 10). vocal center originated in the SVZ. Although the issue of

cell migration needs further confirmation using in vitroexperimentation, our cell migration data are in agreement

4 .1. Effects of lesions on the SVZ and the with the findings of Scharff et al. [40] regarding the SVZmicroenvironment of the lesion site contribution to adult neurogenesis. Our study extended

their basic findings to a region of the adult brain that is notThe impact of bilateral electrolytic lesions is reflected normally neurogenic.

throughout the whole brain. Changes in the SVZ and the The combined evidence suggests that lesion-induced cellmorphological changes at the lesion site are immediate and proliferative activities in the subventricular zone and thedramatic. Successive morphological changes at the lesion concurrent morphological cell changes in the hypo-site, from apoptotic cells to the transformation of the thalamus are all related to the recruitment of newborn

90 J. Cao et al. / Brain Research 943 (2002) 80 –92

neurons in the adult hypothalamus. Alternatively, the 4 .4. Social factors and survival of neuronsnewborn neurons we observed might be in situ differentia-tion of glia, given that astrocytes can behave as neuronal Given that neuronal recruitment in the adult hypo-stem cells in vitro [5]. It seems unlikely however because thalamus is not pre-programmed, survival of the newbornin situ neurogenesis involves a much shorter time frame as neurons may be more critically dependent on other suppor-shown in the study by Magavi et al. [29]. It is intriguing tive factors.that the level of non-neuronal proliferated cells at the Several recent studies on adult hippocampal systems inlesion site remains high 5 months after lesions. Inves- rats suggest that survival of new neurons depends ontigation of the role of these reactive astrocytes as cellular whether they were actively recruited for specific functions.cues to biological function [39] should throw some insight Thus, running [46] and specific learning tasks [19] pro-on understanding the dynamics of cellular changes after mote neurogenesis and neuronal survival in the adultbrain damage. mouse hippocampus. Likewise, in songbirds, singing en-

hances new neuronal survival in the adult high vocal center[25]. In the present study, birds were kept in individual

4 .2. Specificity of markers used in the study cages, a destitute condition that does not allow the bird toengage in VMN mediated behavior, such as courtship. It

The antibodies against 3A7 do not distinguish between may be argued, therefore, that in a socially enrichedtanycytes and radial glia. Both the tanycytes and radial environment (e.g. housed with a receptive female), theglial cells express cytoskeletal proteins that play some newly generated neurons would have a better chance ofroles in axonal guidance, transport, and support [9,36,37]. surviving. In fact, we have previously shown that VMNIn the adult rat mediobasal hypothalamus, the tanycytes lesioned males had a statistically greater chance to recoverwere shown to support the regeneration of monoaminergic a full repertoire of courtship behavior when housed with aaxons [10]. Interpretation of our data is based on the female than in a solitary environment [7]. If new neuronsfinding that in the adult songbird, 3A7 preferentially stains were involved in recovery of behavior, we expect that aguiding glial fibers that support neuronal migration [17]. male housed with a female would have more newborn

We used antibodies against Hu to identify neuronal neurons surviving than a male in solitude. This work isprecursor cells [6,30,47]. To identify differentiated neu- currently in progress in our laboratory.rons, we used both Hu and antibodies against NeuN. Usingthe same two neuron-specific markers (Hu and NeuN),Magavi et al. [29] reported cell death induction of neuro- 4 .5. Comparison with songbirdsgenesis in the neocortex of adult mice.

In the study by Scharff et al. [40], specific classes ofHVC neurons were selectively lesioned. The subset of

4 .3. Retroactive effect of lesions on proliferative activity HVC neurons that project to RA was replaced but not thesubset of HVC neurons that project to area X. Since the

We used BrdU to identify cells born before lesion in subset of HVC-X neurons does not undergo adult turnoverboth short- and long-term studies. A comparison of the in the intact animal, this suggests that apoptotic death-

1number of BrdU cells between the lesioned and control triggered neuronal replacement is highly selective. Ourbrains of both age groups (Fig. 3) indicates that there were findings in the adult hypothalamus, however, suggest that

1more BrdU cells in the lesioned than in control brains. It neuronal replacement can occur in brain areas that do notappears that electrolytic lesion may have retroactively normally undergo turnover. In the present study, the outerfacilitated the proliferative activity of progenitor cells born area of the lesion site expressed radial glia-like fibers thatbefore the lesion. Alternatively, an increased number of potentially provided a directed guide for new neurons to

1BrdU cells in the lesioned brain may reflect enhanced the damaged area. Since microenvironmental changes weresurvival of the prelesion newborn cells. Enhanced survival not monitored in the study by Scharff et al., we could notof the prelesion newborn would mean a lower amount of rule out the possibility that differences in microenviron-apoptosis in the lesioned than non-lesioned birds. This was ment may account for the discrepancy. Systematic com-not the case: lesions did not have any effect on the rate of parisons of apoptosis, microenvironmental changes and

1apoptosis in SVZ. The increased number of BrdU cells in optimal stimulation may provide some insight to thethe lesioned brains, therefore, was more likely a result of question as to why some neurons are replaced and some

1an increased rate of cell proliferation. How many BrdU are not.1 1cells were double-labeled with Hu or NeuN ? Due to In summary, adult brains of mammals and birds are not

1 1proximity of colors, light brown (Hu , NeuN ) vs. dark generally endowed with mechanisms for cell repair. How-1brown (BrdU ), it was difficult to make accurate count of ever, we have shown that lesion and/or lesion-induced cell

these double-labeled cells and we did not include them in loss facilitated cell proliferative activity in the subventricu-our analysis. lar zone, and concurrently induced morphological changes

J. Cao et al. / Brain Research 943 (2002) 80 –92 91

neurons upon ependymally derived radial guide cells in explant(expression of radial glial like fibers) which may provide acultures of the adult songbird forebrain, Glia 8 (1993) 150–160.favorable environment for local neuron recruitment.

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