Research ArticleThe Cell Birth Marker BrdU Does Not Affect Recruitment ofSubsequent Cell Divisions in the Adult Avian Brain

Anat Cattan1 Amir Ayali12 and Anat Barnea3

1Department of Zoology Tel Aviv University 6997801 Tel Aviv Israel2Sagol School of Neuroscience Tel Aviv University 6997801 Tel Aviv Israel3Department of Natural and Life Sciences The Open University of Israel 108 Ravotsky Street PO Box 808 43107 Rarsquoanana Israel

Correspondence should be addressed to Anat Barnea anatbaopenuacil

Received 10 June 2014 Accepted 26 October 2014

Academic Editor Giovanni Scapagnini

Copyright copy 2015 Anat Cattan et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

BrdU is commonly used to quantify neurogenesis but also causes mutation and has mitogenic transcriptional and translationaleffects In mammalian studies attention had been given to its dosage but in birds such examination was not conducted Ourprevious study suggested that BrdUmight affect subsequent cell divisions and neuronal recruitment in the brain Furthermore thiseffect seemed to increase with time from treatment Accordingly we examined whether BrdUmight alter neurogenesis in the adultavian brain We compared recruitment of [3H]-thymidine+ neurons in brains of zebra finches (Taeniopygia guttata) when no BrdUwas involved andwhen BrdUwas given 1 or 3months prior to [3H]-thymidine In nidopallium caudale HVC and hippocampus nodifferences were found between groups in densities and percentages of [3H]-thymidine+ neurons The number of silver grains per[3H]-thymidine+ neuronal nucleus and their distribution were similar across groups Additionally time did not affect the resultsThe results indicate that the commonly used dosage of BrdU in birds has no long-term effects on subsequent cell divisions andneuronal recruitment This conclusion is also important in neuronal replacement experiments where BrdU and another cell birthmarker are given with relatively long intervals between them

1 Introduction

DNA replication of stem cells during the S-phase can beused to birth-date proliferating cells by supplying exogenousmarkers that are incorporated into the DNA of the cells Themarker which is retained in the postmitotic cell enables thesubsequent detection of a cohort of dividing cells that werelabeled at a known time and therefore provides a tool bywhich to quantify neurogenesis the effect of various condi-tions on it and the fate of the new neurons at a later date(reviewed in [1])

The traditional approach to detection of cell prolifera-tion is by incorporation of [3H]-thymidine using autora-diography [2] This technique has been used for decadesto investigate various processes in the CNS (eg [3ndash6])and has been invaluable for the study of the time oforigin of neurons in various species including birds [1]rodents [7] and nonhuman primates [8 9] However due to

the radioactivity of [3H]-thymidine and the relatively longperiod which is required for autoradiography over the yearsanalogs have been introduced as an alternative of which themost commonly used to date is 5-bromo-21015840-deoxyuridine(BrdU) [10] Because [3H]-thymidine and BrdU label cells foronly about 2 h after injection [11] and perhaps for an evenshorter time in birds [12] the time of cell division can bedetermined Both markers have advantages and drawbacks(reviewed in [1]) and hence are preferable for some applica-tions and limited for others One advantage of BrdU is that itcan be quickly detected by immunocytochemistry Moreoverit can be used in combination with immunoperoxidaselabeling for phenotypic markers which can then be detectedby bright-field fluorescence and confocal microscopy todetermine colocalization (eg [13])

A disadvantage of BrdU is that its molecular structureinherently differs from the natural structure of thymidinecausing steric hindrance (Figure 1) Consequently BrdU is

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 126078 11 pageshttpdxdoiorg1011552015126078

2 BioMed Research International

HO

OH

O

OO

NH

N

C3H3

-Thymidine[3H]

(a)

Thymidine

HO

OH

O

OO

NH

N

(b)

BrdU

Br

HO

OH

O

OO

NH

N

(c)

Figure 1 Molecular structures of (a) [3H]-thymidine (b) thymidine (c) BrdU

a toxic mutagenic substance and can cause abnormalities inDNA transcription and protein translation which could leadtomutation and cell toxicity compromising the overall healthand behavior of the organisms both in fetal and in postde-velopmental stages [14ndash16] In addition BrdU triggers celldeath and the formation of teratomas alters DNA stabilitylengthens cell cycle and has mitogenic transcriptional andtranslational effects on the cells that incorporate it (reviewedin [17]) The delaying effect of BrdU on DNA synthesis andcell division is probably caused by structural changes in theDNA helixes which occur when a large amount of BrdU isincorporated into both strands of theDNA [18] FurthermoreBrdU has been found to cause abnormal proliferation invivo [19] although others found no cytotoxic effects on adultneurogenesis [20] In vitro it has been shown to be toxicto neuronal precursors at the concentrations used in mostlabeling studies [21]

Since the evidence indicates that BrdU might affect thestudied cell population when its use as a birth-date markerwas established much attention was given to its dosage andthe frequency of exposure in order to minimize toxicityand avoid misleading results (eg [10 11 22ndash27]) In adultmammals a dose of 50mgkg given daily for up to 12 days anddoses of up to 300mgkg did not cause adverse physiologicaleffects or toxic effects in dividing cells in the dentate gyrus[11 28] However it is possible that BrdU is less toxic inadults than in fetuses or juveniles Whereas all these studieswere performed on mammals in birds such a thoroughexamination has not been conducted Instead BrdU dosagein avian research is similar to that used in fetal mammalsbecause of the similar high metabolic rates of the two groupsThus in birds commonly used doses are sim60ndash100mgkgbody mass (eg [29ndash31])

In one of our previous studies we encountered surprisingresults in a study with zebra finches (Taeniopygia guttata)To provide evidence for neuronal replacement we used twocell birth markers in the same birds BrdU to label neuronsborn 1 or 3 months prior to social manipulation [32] and[3H]-thymidine to label neurons born during the 5 daysimmediately before themanipulation (unpublished data)Wehypothesized that if the total number of neurons remainsconstant then as the BrdU+ neurons decreased the [3H]-thymidine+ ones would increase Since in previous studieswith the same setup [33 34] we had already labeled neurons

that had been born immediately prior to social manipulationwith [3H]-thymidine we expected similar patterns of [3H]-thymidine+ neurons to appear also in the recent study butthis was not the case Previously more neurons had beenrecruited if birds were placed in a complex social settingthan if they were kept in a simple one However this timeno significant differences were found between experimentalgroups and recruitment levels were lower and similar tothose previously observed in birds that were kept in a simplesocial setting Moreover in our recent study time was afactor in the brain region HVC the longer interval betweenBrdU and [3H]-thymidine treatments (3 months) had asignificantly greater negative effect on recruitment of [3H]-thymidine+ neurons compared with that when the intervalbetween treatments was shorter (1 month)

These unexpected findings raised the possibility thatBrdU might have interfered with the mechanism of neuro-genesis as follows BrdU the first marker used incorporatedinto dividing VZ mother cells these gave rise to BrdU+neuronswhichmigrated to their final destination in the brainwhere we recorded them However BrdU incorporation intothe mother cells might have altered them and by doing sonegatively affected their ability to divide again and producemore neurons If so then this effect was then reflected in theunexpected results that we obtained when we later treatedthe same birds with the second birth-date marker [3H]-thymidine and recorded [3H]-thymidine+ neurons In otherwords we allowed for the possibility that BrdU incorporationinto dividing VZ stem cells might block or change theirability to divide again and give rise to more daughter cellsAs a result fewer mother cells would produce new neuronsin the future and this would affect the number of neuronsavailable for recruitment in the brain Under such a scenariothe experimental tool that we applied may have interferedwith the mechanism that it was intended to investigate

Accordingly the aim of this study was to address thepossibility which has not been tested before that BrdU usedas a research tool might alter neurogenesis in the adultavian brain To test this we compared the results of [3H]-thymidine labeling when no BrdUwas involved (control) andwhen BrdU was given 1 or 3 months prior to [3H]-thymidinetreatment If BrdU interferes with subsequent divisions ofthe same cells then the control will yield more [3H]-thymidine+ neurons than in the other groups Moreover

BioMed Research International 3

2Age (month)

0(hatching)

4 76

Breeding colony

Control

3M

1M

1sttreatment salineBrdU

Standard cage Aviary

Saline

BrdU

~83

Perfusion

Location

Saline

Saline

Saline

BrdU

Treatment

2ndtreatment

salineBrdU

3rdtreatment

[3H]

[3H]

[3H]

[3H]

Figure 2 A schematic illustration of the experimental design and groups See text for details

if time negatively affects the process then a 1-month intervalbetween treatmentswill yieldmore [3H]-thymidine+ neuronsthan a 3-month interval

Several studies already used two birth-datemarkers in thesame animal in order to label neuronal subpopulations of dif-ferent ages and to reveal the relationships between these sub-populations in response to specific conditions (reviewed byLlorens-Martın andTrejo [35]) All these studies were done inmammals and many of them used other thymidine analogsIdU and CldU with relatively short intervals between them(hours to a fewweeks) However to the best of our knowledgeour study is the first to test whether the marker which isused first might interfere with subsequent cell divisions andaffect the outcome of a second marker which is used in thesame animal after relatively long intervals More specificallywe tested the validity of using BrdU as the first markerin light of the evidence that indicates its possible toxicity(see above) Other studies have used BrdU as a single markeror combined it with another one simultaneously or at shortintervals (eg [10 11]) Our experimental design was thusintended to indicate whether BrdU has a long-term effect onthe future functionality of neurogenesis This can be highlyrelevant to experiments which examine the dynamics ofneuronal replacement by using two birth markers

2 Materials and Methods

21 Experimental Design As noted in the Introductionthe present study derives from a previous one from ourlaboratory [32] and therefore the experimental design andmethods are similar to those described there A schematicpresentation of the experimental design and groups is pre-sented in Figure 2 The study was approved by the Tel AvivUniversity Institutional Animal Care and Use Committee(permit L-09-003) and was carried out in accordance withits regulations and guidelines regarding the care and use ofanimals for experimental procedures Food and water were

provided ad libitum and consisted of millet flies maggotsgreen vegetables and crushed hard boiled eggs

Male zebra finches (Taeniopygia guttata) were reared inoutdoor breeding colonies at the I Meier Segals Garden forZoological Research at Tel Aviv University and banded withnumbered plastic rings for individual identification At theage of twomonths when experimentalmales (ExMs) becomeindependent they were removed from their native colonyrandomly allocated to an experimental group and housedoutdoors in a standard cage (68 times 345 times 455 cm) with threeother unrelated adult individuals (one female and twomales)This was done to avoid the stress that in this very socialspecies might result from isolation Each cage was visuallyisolated from the outside environment and placed far enoughfrom other cages to achieve auditory isolation

The ExMs remained in this setting until they were aboutfour months old According to their designated experimentalgroup each ExM then received the first treatment of fivedaily intramuscular injections of either 130 120583L saline (NaCl09 as a sham treatment) or 130 120583L (ie 100mgkg) of thecellular birth-date marker 5-bromo-21015840-deoxyuridine (BrdUSigmaUltra diluted 10mgmL in sterile water Sigma) ThisBrdU dose is similar to that used by others to study adultneurogenesis in birds (eg [29ndash32]) The ExMs were keptunder the above conditions for two more months whenthey received a second treatment of five daily intramuscularinjections of either 130 120583L saline or 130 120583L BrdU accordingto their experimental group After an additional month allExMs received a third treatment of five daily intramuscularinjections of 50 120583L of a radioactive form of thymidine(PerkinElmer NET-027 [3H]-thymidine 5mCi) which is amarker of DNA synthesis and therefore of cell birth Twohours after the last [3H]-thymidine injection each ExM wasmoved to a large outdoor aviary (15 times 15 times 2m) where itencountered a complex social environment with a preexistinggroup of 40ndash45 adults of both sexes all of them strangers tothe ExM Birds in any given aviary could neither hear nor see

4 BioMed Research International

those in the other aviaries The ExMs remained in the newsocial environments for 40 d until they were sacrificed

All cages and aviaries were exposed to natural conditions(101ndash147 h light per day and mean 12ndash30∘C daily temper-ature) Our birds are able to breed any time of the yearunder these conditions Therefore and because the ExMs foreach experimental group were obtained at all times of theyear seasonal changes in temperature and photoperiod wereunlikely to have affected the outcome of our study

To recapitulate in the control group (119899 = 7) ExMsreceived saline injections in the first and second treatmentsfollowed by [3H]-thymidine injections in the third treatmentand were then transferred to a new social setting Hencein this group there was no BrdU treatment In group 1M(1 month 119899 = 7) ExMs received saline injections in thefirst treatment and BrdU injections in the second treatmentfollowed by [3H]-thymidine injections in the third treatmentand then were transferred to a new social setting Hence inthis group there was a one-month interval between BrdU and[3H]-thymidine treatments In group 3M (3 months 119899 = 6)ExMs received BrdU injections in the first treatment andsaline injections in the second treatment followed by [3H]-thymidine injections in the third treatment and were trans-ferred to a new social setting Hence in this group there wasa three-month interval between BrdU and [3H]-thymidinetreatments

ExMs were sacrificed 40 d after being introduced intotheir new social setting Previous work with another song-bird the canary (Serinus canaria) showed that neuronsborn in adulthood can take from 8 d [36] to 30ndash40 d [37]to reach their final destination in the telencephalon Thusin all groups we probably recorded labeled neurons thathad already reached their final destination The commondenominator among all groups is that [3H]-thymidine wasgiven prior to the social change while BrdU was giveneither one or three months before [3H]-thymidine and socialchange Thus the results can show whether BrdU affectssubsequent neurogenesis (reflected by the number of [3H]-thymidine+ neurons in the various groups) and if so whethertime plays a role in this process

22 Body and Brain Mass Body mass was used as indirectevidence for overall health and recorded four times on thefirst day of each of the three treatments and at the end ofthe experiment At the end of the experiment followingperfusion (see below) brains were weighed

23 Histology Immunohistochemistry and AutoradiographyForty days after the last [3H]-thymidine injection ExMswere weighed sacrificed with an overdose of anesthesia(006mL of Ketalar diluted 10 times followed by 006mLof xylazine) and fixed with an intracardiac perfusion withsaline followed by 4 paraformaldehyde in 01M phosphatebuffer (PB pH 74) Brains were removed weighed andtransferred into PB at 4∘C After 1-2 d brains were dehydratedin alcohols embedded in polyethylene glycol blocked andcut transversely at 6 120583m intervals

Two sets of serial sections (at intervals of 120120583m) weremounted on slides (Superfrost PLUS) using a solution of 01

10120583m

Figure 3 A new neuron (marked with an arrow) labeled with [3H]-thymidine (expressed by the silver grains) and stained with anti-Hu(brown)

BSA (albumin bovine minimum 98) in PBS Set A wasstained to enable [3H]-thymidine+ neurons identificationSince our main goal was to determine whether an earlierexposure to BrdU interferes with future cell divisions set Awas used to obtain the data to answer this question We fol-lowed a previous protocol from our laboratory (for details see[32]) In brief sections were incubated with anti-HuCHuDmouse IgG

2b a primary antibody that is affixed specificallyto neurons [38] and then with DAB which stained theneurons in the tissue brown Sections then went throughautoradiography and were developed after 4 weeks Thisprocedure yielded neurons that were stained brown and new[3H]-thymidine+ neurons that were brown with silver grains(Figure 3) Set B was stained for the identification of BrdU+neurons and was only used to validate the BrdU treatmentnamely to ensure that BrdU had been incorporated into thedividing cells prior to the thymidine treatment For this setwe used an immunohistochemical procedure that had beenpreviously applied in our laboratory (for details see [32]) andis similar to that which had been used by others [30] Thisprocedure yielded neurons that were stained brown and newBrdU+ neurons that were brown with fluorescent red nuclei(Figure 4)

24 Mapping and Quantification We counted new [3H]-thymidine+ neurons in three brain regions nidopallium cau-dale (NC) HVC and hippocampus (HC) (Figure 5(a)) TheNC was chosen because it includes auditory relays that prob-ably play a role in vocal communication and in the processingof other types of auditory inputs [39 40] The HVC is part ofthe song control system and is also involved in vocal com-munication (reviewed by [41]) HC is known to be involvedin the processing of spatial information [42] These brainregions were previously investigated in our laboratory andneuronal recruitment in them was affected by social manip-ulation [32 34 43] In addition these brain regions haveboundaries that are easy to recognize in transverse sectionsfor the NC and HVC we followed the criteria described by[33] and for the HC those described by [34]

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10120583m

(a) (b) (c)

Figure 4 Three confocal images of the same brain tissue demonstrating double-labeling of Hu (brown) and BrdU (fluorescence) Two (rednuclei) of the several neurons (Hu+ brown) shown in this field were born at the time of BrdU injections (a) BrdU labeling (b) Hu labeling(c) Colocalization of BrdU and Hu labeling

The NC stretches rostrocaudally over a distance of1200120583m along which we sampled five sections The middlesection went through the robust nucleus of arcopallium (RA)at its largest diameter (P12 in the canary atlas [44]) Twomore sections were obtained rostrally and caudally respec-tively at distances of 360 and 600120583m from the middle one(Figure 5(b)) In the HC which stretches over 2000120583m inthe rostrocaudal axis we sampled five sections per brainThe most rostral section corresponded to A40 in the canaryatlas [44] and the fifth and most caudal one corresponded toA02 there The three other in-between sections were evenlydistributed so that on average the distance between eachmapped section and the next one was 480 120583m (Figure 5(c))In the HVC which is 240ndash720120583m along its rostrocaudal axiswe sampled all mounted sections in which it appeared (five toseven per brain) Rostrally the first section corresponded toAP00 in the canary atlas [44] (Figure 5(d))

We used a computerized brain-mapping system (Neu-rolucida Stereo Investigator Micro-BrightField) to draw theboundaries of theNCHVC andHC in each section sampledenter the position of [3H]-thymidine+ neurons count totalneuronal densities and quantify other neuronal parameters(see below) All mapping was done ldquoblindrdquo as to the exper-imental conditions and by using a 63x objective Previouswork in our laboratory [32] had revealed no hemisphericdifferences in all regions in the number of labeled neurons permm3 Therefore we mapped sections only from the righthemisphere

As the NC is a large brain region after conducting apreliminary evaluation we scanned about 35 of the wholesections by using the fractionator probe in our mappingsystem However in the HVC and HC which are relativelysmall we completely scanned all the mapped sections usingthe meander scan probe In the HVC and HC we alsomeasured the neuronal nuclear diameters of all of the newneurons Since the NC is a large region with hundreds ofnew neurons per section we measured neuronal nucleardiameters only in the fifth section in which we sampledsix nonoverlapping squares (each of an area of 19600120583m2)randomly chosen by the software and which yielded at leastten [3H]-thymidine+ neurons

From the knowledge of section thickness and mean neu-ronal nuclear diameter of labeled neurons in a certain brain

region we used the Abercrombie stereological correction[45] to estimate the number of [3H]-thymidine+ neurons permm3 in each brain region In the NC and HC these estimateswere obtained for each section In the HVC data from allsections mapped for each brain were pooled with each brainrepresented by a single estimate of the number of [3H]-thymidine+ neurons per mm3 This was done because of thevariability in the number of sections of this region in eachbrain which prevented our comparison of them

In each brain region we also estimated the density of totalneurons ([3H]-thymidine+ and unlabeled ones) per mm3This was done in the fifth section in which all neurons werecounted in six squares as described above for the neuronalnuclear diameter of new neurons In the first and last squareswe also measured nuclear diameters of all neurons ([3H]-thymidine+ and unlabeled ones) As explained above thisallowed us to estimate the total number of neurons per mm3in each brain region and also to calculate the percentage ofnew neurons per mm3 out of the total number of neurons

25 Statistical Analysis Most of the statistical analysis wasperformed using STATISTICAcopy 8 (StatSoft Inc) Analyzingthe interaction between sections in the parameter of numberof [3H]-thymidine+ neurons per mm3 in HC was performedusing SPSS v15 with ANOVA repeated-measures within-subjects contrasts All data expressed as number of neuronsper mm3 or proportions were transformed before the statis-tical analysis using the square root transformation (Thesekinds of data number of discrete elements per unit tend tohave a poison distribution and the suitable transformationfor such a case is the square root transformation [46])119875 lt 005 was considered significant throughout To comparedifferent parameters obtained from several section levelsanalysis of variance was performed using Two-Way ANOVA(repeated-measures) Since we had only one uniform discretecategorical variable (number of months between exposureto BrdU and [3H]-thymidine) all ANOVA tests used thefixed-effects model (model 1) To analyze the differences inbody mass between experimental groups we used Two-WayANOVA (repeated-measures) in model 1 To analyze thedifferences in brainmasswe usedOne-WayANOVA inmodel1 [46]

6 BioMed Research International

R C

NCHVCHC

2mm

(a)

D

L M

V

(b)

D

L M

V

(c)

D

L M

V

(d)

Figure 5 Schematic views of the three investigated brain regions (a) Top view of the brain rostral section is to the left and caudal sectionis to the right We indicate the range within which frontal sections were taken from the nidopallium caudale (NC) hippocampus (HC) andHVC Several sections were sampled along the rostrocaudal axis of each brain region (for details see text) only three sections are shown herethe most rostral the middle and the most caudal (from left to right) in NC (b) HC (c) and HVC (d) Cerebellum Cb lamina arcopallialisdorsalis LAD lateral ventricle V nidopallium N nucleus robustus arcopallii RA and tectum opticum TeO Orientations dorsal D lateralL ventral V and medial M adapted from [43] and illustrated by A Cattan

BioMed Research International 7

0

05

10

15

20

25

30

NC-

cont

rol

NC-

1M

NC-

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-con

trol

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-1M

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-3M

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rol

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1M

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Mea

n (plusmn

SE) d

ensit

y of

-th

ymid

ine

labe

led

neur

ons (

neur

ons

mm

3)

times103

[3H]

Figure 6Mean (plusmnSE) densities (neuronsmm3) of [3H]-thymidine+neurons in NC HVC and HC in the three experimental groups(control 1M one month after BrdU treatment 3M three monthsafter BrdU treatment) In all the three investigated brain regionsno significant differences were found between experimental groupsSample sizes control and 1M = 7 brains 3M = 6 brains

3 Results

The main aim of this study was to determine whethertreatment with the cell birth marker BrdU affects subse-quent cell divisions and the recruitment of neurons thatdifferentiate from these divisions into various brain regionsWe thus injected birds with BrdU and either one or threemonths later treated them with another cell birth marker[3H]-thymidine (groups 1M and 3M resp) A controlgroup (group C) received only [3H]-thymidine treatmentTo answer our question we then compared the number of[3H]-thymidine+ neurons in the tested brain regions betweenthe three experimental groups Our prediction was that ifBrdU affects subsequent cell divisions and the recruitmentof neurons that differentiate from these divisions then fewer[3H]-thymidine+ neurons will be found in the two groupsthat were treated with BrdU (1M and 3M) compared to thecontrol In addition if the effect of BrdU is time-dependentwe expected fewer [3H]-thymidine+ neurons in group 1Mcompared to group 3M

31 Densities of [3H]-Thymidine+ Neurons In the three inves-tigated brain regions we found no significant differencesbetween the experimental groups in the density of [3H]-thymidine+ neurons (Figure 6 NC F

(217)= 10508 119875 =

0371 HVC F(217)= 03399 119875 = 0716 HC F

(217)=

11787 119875 = 0331) This indicates that BrdU does not affectsubsequent cell divisions and the recruitment of neurons thatdifferentiate from these divisions This holds true both forthe subsequent cell divisions which occur relatively soon (1month) after BrdU administration and for divisions whichoccur after longer intervals (3 months)

In addition in the NC no significant differences werefound between sections (F

(468)= 16420 119875 = 0173) and

00020406081012141618202224

Mea

n

(plusmnSE

)-th

ymid

ine l

abel

edne

uron

s out

of t

otal

neu

rons

mm

3

NC-

cont

rol

NC-

1M

NC-

3M

HVC

-con

trol

HVC

-1M

HVC

-3M

HC-

cont

rol

HC-

1M

HC-

3M

[3H]

Figure 7 Mean (plusmnSE) percentages () of [3H]-thymidine+ neuronsin NC HVC and HC in the three experimental groups (control1M one month after BrdU treatment 3M three months after BrdUtreatment) In all the three investigated brain regions no significantdifferences were found between experimental groups Sample sizescontrol and 1M = 7 brains 3M = 6 brains

no significant interaction was found between sections andgroups (F

(868)= 07481 119875 = 0649) and accordingly

the overall mean density in this region was 1940 plusmn 132[3H]-thymidine+ neuronsmm3 In the HVC no significantdifferences were found between groups (F

(217)= 03399 119875 =

0716) and accordingly the overall mean density was 2187plusmn194 [3H]-thymidine+ neuronsmm3 In the HC significantdifferences were found between sections (F

(468)= 75078

119875 lt 0001) but no interaction was found between sectionsand experimental groups (F

(868)= 06892 119875 = 0699)

and accordingly the overall mean density in this region was232 plusmn 26 [3H]-thymidine+ neuronsmm3

32 Percentages of [3H]-Thymidine+ Neurons A similar out-come of no significant differences between experimentalgroups in the three investigated brain regions was alsoobtainedwhenwe calculated percentages of [3H]-thymidine+neurons (Figure 7 NC F

(217)= 08405 119875 = 0448 HVC

F(217)= 00692 119875 = 0933 HC F

(217)= 09245 119875 = 0415)

This indicates again that BrdU does not affect subsequent celldivisions and the recruitment of neurons that differentiatefrom these divisions both for divisions which occur relativelysoon (1 month) after BrdU administration and for divisionswhich occur after a longer interval (3 months)

In addition in the NC no significant differences werefound between sections (F

(468)= 15705 119875 = 0192) and

no significant interaction was found between sections andgroups (F

(868)= 07614 119875 = 0637) accordingly the overall

mean percentage of [3H]-thymidine+ neurons in this regionwas 16 plusmn 06 In the HVC the overall mean percentageof [3H]-thymidine+ neurons was 13 plusmn 01 In the HCa significant difference was found between sections (F

(468)=

77432 119875 lt 0001) but since no interaction was foundbetween sections and groups (F

(868)= 06902 119875 = 0698)

8 BioMed Research International

00

01

02

03

04

05

2ndash4

5ndash7

8ndash10

11ndash1

314

ndash16

17ndash1

920

ndash22

23ndash2

526

ndash28

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132

ndash34

Ove

r 35

2ndash4

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11ndash1

314

ndash16

17ndash1

920

ndash22

23ndash2

526

ndash28

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ndash34

Ove

r 35

2ndash4

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314

ndash16

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920

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526

ndash28

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132

ndash34

Ove

r 35

Number of silver grains Number of silver grainsNumber of silver grains

Neu

rons

out

of t

otal

()

NC HVC HC

Figure 8 Distributions of the number of exposed silver grains in [3H]-thymidine+ neurons in NC HVC and HC in brains of birds fromthe control group (Q 119899 = 7) 1M (one month after BrdU treatment ◻ 119899 = 7) and 3M (three months after BrdU treatment 998771 119899 = 6)

we pooled the results from all sections in each brain andthe overall mean percentage of [3H]-thymidine+ neurons was02 plusmn 002

33 Nuclear Diameter of [3H]-Thymidine+ Neurons No sig-nificant differences between groups were found in meannuclear diameter of [3H]-thymidine+ neurons in either theNC (F

(217)= 01193 119875 = 0888) or the HC (F

(215)= 06914

119875 = 0516) Accordingly overall mean nuclear diameterof NC neurons was 147 plusmn 03 120583m (119899 = 283 neurons outof 20 brains) and of HC neurons 146 plusmn 02 120583m (119899 = 241neurons out of 20 brains) This finding further supports theindication that BrdUdoes not affect subsequent cell divisionsHowever in the HVC significant differences (F

(217)=

45969 119875 = 0025) were observed between groups in themean nuclear diameter of [3H]-thymidine+ neurons In thecontrol group HVC neurons were significantly bigger thanthose in groups 1M (df = 17 119875 = 0043) and 3M (df = 17119875 = 0049) Accordingly mean [3H]-thymidine+ neuronalnuclear diameter in the control group was 149 plusmn 04 120583m(119899 = 333 neurons out of 7 brains) in group 1M 134 plusmn 03 120583m(119899 = 392 neurons out of 7 brains) and in group 3M 134 plusmn06 120583m (119899 = 364 neurons out of 6 brains)

34 Number of Exposed Silver Grains per [3H]-Thymidine+Neuronal Nucleus and Their Distribution In all three inves-tigated brain regions no significant differences were foundbetween groups in themean number of silver grains per [3H]-thymidine+ neuronal nucleus (NC F

(217)= 00363 119875 =

0964 HVC F(217)= 07189 119875 = 0501 HC F

(217)=

10407 119875 = 0374) This indicates that in each brain regionthe [3H]-thymidine+ neurons derive from the same originalpopulation and undergo the same number of divisionsDistributions of the number of exposed silver grains in [3H]-thymidine+ neuronal nuclei further support this conclusion(Figure 8)

35 Brain and Body Mass No significant differences werefound in body mass between experimental groups (F

(218)=

13651 119875 = 0280) as well as between experimental stages

(F(354)= 27264 119875 = 0052) with no interaction between

groups and stages (F(654)= 08207 119875 = 0558) Accordingly

mean body mass was 134 plusmn 08 g (119899 = 27) Similarly nosignificant differences were found in brain mass betweenexperimental groups (F

(218)= 00701 119875 = 0932) and mean

brain mass was 047 plusmn 002 g (119899 = 21)

4 Discussion

Our data indicate that BrdU used as a research tool in theadult avian brain does not affect the recruitment of neuronsborn after its use in subsequent cell divisions in the VZWhile we are very much aware of the caution one should takewhen accepting a null hypothesis based on nonsignificantresults we feel that in this case our conclusion is supported byseveral semioverlapped as well as independent results Firstlyno significant differences were found between the control andexperimental groups in levels of neuronal recruitment in thethree investigated brain regions (NC HVC and HC) testedindependently This holds true both for densities of [3H]-thymidine+ neurons per mm3 and for percentages of [3H]-thymidine+ neurons out of the total neuronal populationsMoreover time does not affect this outcome as evident fromthe similar results obtained when we recorded recruitmentof neurons that originated from subsequent cell divisionsrelatively soon after BrdU administration (1month) and fromdivisions that occurred after a longer interval (3 months)

The conclusion that BrdU treatment in the avian braindoes not affect subsequent cell divisions in the VZ and theneurons that differentiate from these divisions draws furthersupport from the finding that in most cases there were nogroup differences in the mean nuclear diameter of [3H]-thymidine+ neurons (apart from HVC in which nucleardiameters were larger in the experimental groups comparedto the control) Neuronal nuclear diameter decreases withage [36 47] and therefore can be used as an indicator forneuronal age Since mean neuronal nuclear diameters weresimilar across most groups we can assume that neuronalage was also similar and hence that the [3H]-thymidine+neurons were born within the 5 days of treatment

8 BioMed Research International

00

01

02

03

04

05

2ndash4

5ndash7

8ndash10

11ndash1

314

ndash16

17ndash1

920

ndash22

23ndash2

526

ndash28

29ndash3

132

ndash34

Ove

r 35

2ndash4

5ndash7

8ndash10

11ndash1

314

ndash16

17ndash1

920

ndash22

23ndash2

526

ndash28

29ndash3

132

ndash34

Ove

r 35

2ndash4

5ndash7

8ndash10

11ndash1

314

ndash16

17ndash1

920

ndash22

23ndash2

526

ndash28

29ndash3

132

ndash34

Ove

r 35

Number of silver grains Number of silver grainsNumber of silver grains

Neu

rons

out

of t

otal

()

NC HVC HC

Figure 8 Distributions of the number of exposed silver grains in [3H]-thymidine+ neurons in NC HVC and HC in brains of birds fromthe control group (Q 119899 = 7) 1M (one month after BrdU treatment ◻ 119899 = 7) and 3M (three months after BrdU treatment 998771 119899 = 6)

we pooled the results from all sections in each brain andthe overall mean percentage of [3H]-thymidine+ neurons was02 plusmn 002

33 Nuclear Diameter of [3H]-Thymidine+ Neurons No sig-nificant differences between groups were found in meannuclear diameter of [3H]-thymidine+ neurons in either theNC (F

(217)= 01193 119875 = 0888) or the HC (F

(215)= 06914

119875 = 0516) Accordingly overall mean nuclear diameterof NC neurons was 147 plusmn 03 120583m (119899 = 283 neurons outof 20 brains) and of HC neurons 146 plusmn 02 120583m (119899 = 241neurons out of 20 brains) This finding further supports theindication that BrdUdoes not affect subsequent cell divisionsHowever in the HVC significant differences (F

(217)=

45969 119875 = 0025) were observed between groups in themean nuclear diameter of [3H]-thymidine+ neurons In thecontrol group HVC neurons were significantly bigger thanthose in groups 1M (df = 17 119875 = 0043) and 3M (df = 17119875 = 0049) Accordingly mean [3H]-thymidine+ neuronalnuclear diameter in the control group was 149 plusmn 04 120583m(119899 = 333 neurons out of 7 brains) in group 1M 134 plusmn 03 120583m(119899 = 392 neurons out of 7 brains) and in group 3M 134 plusmn06 120583m (119899 = 364 neurons out of 6 brains)

34 Number of Exposed Silver Grains per [3H]-Thymidine+Neuronal Nucleus and Their Distribution In all three inves-tigated brain regions no significant differences were foundbetween groups in themean number of silver grains per [3H]-thymidine+ neuronal nucleus (NC F

(217)= 00363 119875 =

0964 HVC F(217)= 07189 119875 = 0501 HC F

(217)=

10407 119875 = 0374) This indicates that in each brain regionthe [3H]-thymidine+ neurons derive from the same originalpopulation and undergo the same number of divisionsDistributions of the number of exposed silver grains in [3H]-thymidine+ neuronal nuclei further support this conclusion(Figure 8)

35 Brain and Body Mass No significant differences werefound in body mass between experimental groups (F

(218)=

13651 119875 = 0280) as well as between experimental stages

(F(354)= 27264 119875 = 0052) with no interaction between

groups and stages (F(654)= 08207 119875 = 0558) Accordingly

mean body mass was 134 plusmn 08 g (119899 = 27) Similarly nosignificant differences were found in brain mass betweenexperimental groups (F

(218)= 00701 119875 = 0932) and mean

brain mass was 047 plusmn 002 g (119899 = 21)

4 Discussion

Our data indicate that BrdU used as a research tool in theadult avian brain does not affect the recruitment of neuronsborn after its use in subsequent cell divisions in the VZWhile we are very much aware of the caution one should takewhen accepting a null hypothesis based on nonsignificantresults we feel that in this case our conclusion is supported byseveral semioverlapped as well as independent results Firstlyno significant differences were found between the control andexperimental groups in levels of neuronal recruitment in thethree investigated brain regions (NC HVC and HC) testedindependently This holds true both for densities of [3H]-thymidine+ neurons per mm3 and for percentages of [3H]-thymidine+ neurons out of the total neuronal populationsMoreover time does not affect this outcome as evident fromthe similar results obtained when we recorded recruitmentof neurons that originated from subsequent cell divisionsrelatively soon after BrdU administration (1month) and fromdivisions that occurred after a longer interval (3 months)

The conclusion that BrdU treatment in the avian braindoes not affect subsequent cell divisions in the VZ and theneurons that differentiate from these divisions draws furthersupport from the finding that in most cases there were nogroup differences in the mean nuclear diameter of [3H]-thymidine+ neurons (apart from HVC in which nucleardiameters were larger in the experimental groups comparedto the control) Neuronal nuclear diameter decreases withage [36 47] and therefore can be used as an indicator forneuronal age Since mean neuronal nuclear diameters weresimilar across most groups we can assume that neuronalage was also similar and hence that the [3H]-thymidine+neurons were born within the 5 days of treatment

BioMed Research International 9

Two additional findings further strengthen the conclu-sion that the [3H]-thymidine+ neurons were derived from thesame original population and underwent the same numberof divisions the number of exposed silver grains per [3H]-thymidine+ neuronal nucleus and their distributions werealso similar between groups in all three investigated brainregions This supports our conclusion as the number ofexposed silver grains positively correlates to the amount of[3H]-thymidine incorporated into the nuclei during the S-phase when DNA synthesis occurs If one assumes that allstem cells have a similar duration of S-phase then the ini-tial uptake of [3H]-thymidine+ was presumably comparableacross groups since it occurred when all birds were keptunder the same conditions Under such situation daughtercells that originate from themitotic event that occurred at thetime of treatment are expected to reflect a similar labelingHowever if successive rounds of stem cell divisions takeplace after the initial labeling then a dilution of labelingoccurs each time yielding new generations of neurons withless labeling that is fewer exposed silver grains per labeledneuron [12 48] Hence the number of exposed silver grainsand their distribution in [3H]-thymidine+ neurons enableus to determine whether these neurons derive from thesame population or are the result of several mitotic events[49] Since the number of exposed silver grains and theirdistribution were similar across groups in all the investigatedbrain regions we conclude that the [3H]-thymidine+ neuronswere a result of the samemitotic events and hence that BrdUdid not affect it Finally it is worth noting that body andbrain mass did not differ significantly between experimentalgroups and from this and other observations we infer that allthe birds remained in good health

5 Conclusions

We investigated in the adult avian brain the possibility thatBrdU incorporation into dividing VZ stem cells might blockor change their ability to divide again and give rise to moredaughter cells This is important to know because if sothen using BrdU as a research tool might interfere with themechanism that it was intended to investigate However ourdata indicate that this is not the case since in this study BrdUdid not affect the recruitment of neurons that were born afterits administration Examination of the correct BrdUdosage inbirds has not been done in the past and therefore we believethat our conclusion is relevant to experiments that studyneuronal replacement in avian systems since it confirms thatthe commonly used dosage is acceptable Our study and itsfindings are also novel because to the best of our knowledgeit is the first to test the validity of using BrdU combinedwith another birth-date marker in the same animal withrelatively long intervals between markers Other studies todate have used only one marker or two but simultaneously orat short intervals (eg [10 11]) Therefore the possible effectof BrdU on neurogenesis and its future functionality due toits potential toxicity could not have been detected beforeThe present study which indicates that BrdU does not have along-term effect on the future functionality of neurogenesis

thus has relevancewhen planning experiments which use twobirth markers for example when examining the dynamics ofneuronal replacement

Disclosure

Amir Ayali and Anat Cattan are coauthors

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by Israel Science FoundationGrant 23610 and the Open University Research Fund (AnatBarnea) Thanks are due to the staff of the Meier Segals Gar-den for Zoological Research at Tel Aviv University for theirtechnical help The authors are also grateful to I Gelernterand Dr Rachel Heiblum for their help with statistical analysisand to Naomi Paz for editing the paper

References

[1] A Barnea and V Pravosudov ldquoBirds as a model to study adultneurogenesis bridging evolutionary comparative and neu-roethological approachesrdquo European Journal of Neurosciencevol 34 no 6 pp 884ndash907 2011

[2] R L Sidman I L Miale and N Feder ldquoCell proliferationand migration in the primitive ependymal zone An autoradio-graphic study of histogenesis in the nervous systemrdquo Experi-mental Neurology vol 1 no 4 pp 322ndash333 1959

[3] P Rakic ldquoNeurons in rhesus monkey visual cortex systematicrelation between time of origin and eventual dispositionrdquoScience vol 183 no 4123 pp 425ndash427 1974

[4] J Altman and S A Bayer ldquoMosaic organization of the hip-pocampal neuroepithelium and the multiple germinal sourcesof dentate granule cellsrdquo Journal of Comparative Neurology vol301 no 3 pp 325ndash342 1990

[5] D Crespo B B Stanfield and W M Cowan ldquoEvidence thatlate-generated granule cells do not simply replace earlier formedneurons in the rat dentate gyrusrdquo Experimental Brain Researchvol 62 no 3 pp 541ndash548 1986

[6] A R Schlessinger W M Cowan and D I Gottlieb ldquoAnautoradiographic study of the time of origin and the pattern ofgranule cell migration in the dentate gyrus of the ratrdquo Journal ofComparative Neurology vol 159 no 2 pp 149ndash175 1975

[7] J B Angevine Jr ldquoTime of neuron origin in the hippocampalregion an autoradiographic study in the mouserdquo ExperimentalNeurology vol 11 pp 1ndash70 1965

[8] P Rakic ldquoKinetics of proliferation and latency between final celldivision and onset of differentiation of cerebellar stellate andbasket neuronsrdquo Journal of Comparative Neurology vol 147 no4 pp 523ndash546 1973

[9] P Rakic ldquoLimits of neurogenesis in primatesrdquo Science vol 227no 4690 pp 1054ndash1056 1985

[10] M W Miller and R S Nowakowski ldquoUse of bromodeoxy-uridine-immunohistochemistry to examine the proliferation

10 BioMed Research International

migration and time of origin of cells in the central nervoussystemrdquo Brain Research vol 457 no 1 pp 44ndash52 1988

[11] H A Cameron and R D G Mckay ldquoAdult neurogenesisproduces a large pool of new granule cells in the dentate gyrusrdquoJournal of Comparative Neurology vol 435 no 4 pp 406ndash4172001

[12] A Alvarez-Buylla M Theelen and F Nottebohm ldquoProlifera-tion ldquohot spotsrdquo in adult avian ventricular zone reveal radial celldivisionrdquo Neuron vol 5 no 1 pp 101ndash109 1990

[13] S S Magavi and J D Macklis ldquoIdentification of newborn cellsby BrdU labeling and immunocytochemistry in vivordquo inNeuralStem Cells vol 438 of Methods in Molecular Biology pp 335ndash343 Humana Press 2008

[14] W J Biggers E R Barnea andMK Sanyal ldquoAnomalous neuraldifferentiation induced by 5-bromo-2rsquo-deoxyuridine duringorganogenesis in the ratrdquo Teratology vol 35 no 1 pp 63ndash751987

[15] B Kolb B Pedersen M Ballermann R Gibb and I QWhishaw ldquoEmbryonic and postnatal injections of bromode-oxyuridine produce age- dependent morphological and behav-ioral abnormalitiesrdquo Journal of Neuroscience vol 19 no 6pp 2337ndash2346 1999

[16] J M Barasch and R S Bressler ldquoThe effect of 5-bromod-eoxyuridine on the postnatal development of the rat testisrdquoJournal of Experimental Zoology vol 200 no 1 pp 1ndash8 1977

[17] P Taupin ldquoBrdU immunohistochemistry for studying adultneurogenesis paradigms pitfalls limitations and validationrdquoBrain Research Reviews vol 53 no 1 pp 198ndash214 2007

[18] J C de Almeida H Sauaia and J C Viana ldquo5-Bromo-21015840-deoxyuridine induces visible morphological alteration in theDNA puffs of the anterior salivary gland region of bradysiahygida (Diptera Sciaridae)rdquo Brazilian Journal of Medical andBiological Research vol 43 no 12 pp 1143ndash1152 2010

[19] T L Goldsworthy C S Dunn and J A Popp ldquoDose effectsof bromodeoxyuridine (BRDU) on rodent hepatocyte prolifer-ation measurmentsrdquo Toxicologist vol 12 article 265 1992

[20] A Hancock C Priester E Kidder and J R Keith ldquoDoes 5-bromo-21015840-deoxyuridine (BrdU) disrupt cell proliferation andneuronal maturation in the adult rat hippocampus in vivordquoBehavioural Brain Research vol 199 no 2 pp 218ndash221 2009

[21] M A Caldwell X He and C N Svendsen ldquo5-Bromo-21015840-deoxyuridine is selectively toxic to neuronal precursors invitrordquoEuropean Journal ofNeuroscience vol 22 no 11 pp 2965ndash2970 2005

[22] K A Burns andC-Y Kuan ldquoLow of bromo- and iododeoxyuri-dine produce near-saturation labeling of adult proliferative pop-ulations in the dentate gyrusrdquo European Journal of Neurosciencevol 21 no 3 pp 803ndash807 2005

[23] T L Goldsworthy B E Butterworth and R R MaronpotldquoConcepts labeling procedures and design of cell proliferationstudies relating to carcinogenesisrdquo Environmental Health Per-spectives vol 101 no 5 pp 59ndash65 1993

[24] E Gould and C G Gross ldquoNeurogenesis in adult mammalssome progress and problemsrdquoThe Journal of Neuroscience vol22 no 3 pp 619ndash623 2002

[25] S S Magavi and J D Macklis ldquoIdentification of newborn cellsby BrdU labeling and immunocytochemistry in vivordquoMethodsin Molecular Biology vol 198 pp 283ndash290 2002

[26] G Sekerkova E Ilijic and E Mugnaini ldquoBromodeoxyuridineadministered during neurogenesis of the projection neuronscauses cerebellar defects in ratrdquo Journal of Comparative Neurol-ogy vol 470 no 3 pp 221ndash239 2004

[27] T Takahashi R S Nowakowski and V S Caviness Jr ldquoBUdRas an S-phase marker for quantitative studies of cytokineticbehaviour in the murine cerebral ventricular zonerdquo Journal ofNeurocytology vol 21 no 3 pp 185ndash197 1992

[28] C M Cooper-Kuhn and H Georg Kuhn ldquoIs it all DNA repairmethodological considerations for detecting neurogenesis inthe adult brainrdquo Developmental Brain Research vol 134 no 1-2 pp 13ndash21 2002

[29] N Wang P Hurley C Pytte and J R Kirn ldquoVocal controlneuron incorporation decreases with age in the adult zebrafinchrdquo Journal of Neuroscience vol 22 no 24 pp 10864ndash108702002

[30] LWilbrecht A Crionas and F Nottebohm ldquoExperience affectsrecruitment of new neurons but not adult neuron numberrdquoJournal of Neuroscience vol 22 no 3 pp 825ndash831 2002

[31] C L Pytte M Gerson J Miller and J R Kirn ldquoIncreasingstereotypy in adult zebra finch song correlates with a decliningrate of adult neurogenesisrdquoDevelopmental Neurobiology vol 67no 13 pp 1699ndash1720 2007

[32] E Adar F Nottebohm and A Barnea ldquoThe relationshipbetween nature of social change age and position of newneurons and their survival in adult zebra finch brainrdquoThe Jour-nal of Neuroscience vol 28 no 20 pp 5394ndash5400 2008

[33] D Lipkind F Nottebohm R Rado and A Barnea ldquoSocialchange affects the survival of new neurons in the forebrain ofadult songbirdsrdquo Behavioural Brain Research vol 133 no 1 pp31ndash43 2002

[34] A Barnea A Mishal and F Nottebohm ldquoSocial and spatialchanges induce multiple survival regimes for new neurons intwo regions of the adult brain an anatomical representation oftimerdquo Behavioural Brain Research vol 167 no 1 pp 63ndash742006

[35] M Llorens-Martın and J L Trejo ldquoMultiple birthdating anal-yses in adult neurogenesis A line-up of the usual suspectsrdquoFrontiers in Neuroscience vol 5 article 76 2011

[36] J R Kirn Y Fishman K Sasportas A Alvarez-Buylla and FNottebohm ldquoFate of new neurons in adult canary high vocalcenter during the first 30 days after their formationrdquo Journal ofComparative Neurology vol 411 no 3 pp 487ndash494 1999

[37] A Alvarez-Buylla and F Nottebohm ldquoMigration of youngneurons in adult avian brainrdquoNature vol 335 no 6188 pp 353ndash354 1988

[38] K Barami K Iversen H Furneaux and S A Goldman ldquoHuprotein as an early marker of neuronal phenotypic differ-entiation by subependymal zone cells of the adult songbirdforebrainrdquo Journal of Neurobiology vol 28 no 1 pp 82ndash1011995

[39] G E Vates B M Broome C V Mello and F NottebohmldquoAuditory pathways of caudal telencephalon and their relationto the song system of adult male zebra finches (Taenopygiaguttata)rdquo The Journal of Comparative Neurology vol 366 pp613ndash642 1996

[40] C V Mello G E Vates S Okuhata and F NottebohmldquoDescending auditory pathways in the adult male zebra finch(Taeniopygia guttata)rdquo The Journal of Comparative Neurologyvol 395 no 2 pp 137ndash160 1998

[41] F Nottebohm ldquoWhy are some neurons replaced in adult brainrdquoThe Journal of Neuroscience vol 22 no 3 pp 624ndash628 2002

[42] J R Krebs D F Sherry S D Healy V H Perry and A L Vac-carino ldquoHippocampal specialization of food-storing birdsrdquoPro-ceedings of the National Academy of Sciences of the United Statesof America vol 86 no 4 pp 1388ndash1392 1989

BioMed Research International 11

[43] S Barkan A Ayali F Nottebohm and A Barnea ldquoNeuronalrecruitment in adult zebra finch brain during a reproductivecyclerdquo Developmental Neurobiology vol 67 no 6 pp 687ndash7012007

[44] T M Stokes C M Leonard and F Nottebohm ldquoThe telen-cephalon diencephalon andmesencephalon of the canary Ser-inus canaria in stereotaxic coordinatesrdquo Journal of ComparativeNeurology vol 156 no 3 pp 337ndash374 1974

[45] R W Guillery and K Herrup ldquoQuantification without pontif-ication choosing a method for counting objects in sectionedtissuesrdquo The Journal of Comparative Neurology vol 386 no 1pp 2ndash7 1997

[46] R R Sokal and F J Rohlf Biometry W H Freeman SanFrancisco Calif USA 3rd edition 1995

[47] J R Kirn and F Nottebohm ldquoDirect evidence for loss andreplacement of projection neurons in adult canary brainrdquo TheJournal of Neuroscience vol 13 no 4 pp 1654ndash1663 1993

[48] P K Tekumalla M Tontonoz M A Hesla and J R KirnldquoEffects of excess thyroid hormone on cell death cell prolifer-ation and new neuron incorporation in the adult zebra finchtelencephalonrdquo Journal of Neurobiology vol 51 no 4 pp 323ndash341 2002

[49] S H Hornfeld J Terkel and A Barnea ldquoNeurons recruited inthe nidopallium caudale following changes in social environ-ment derive from the same original populationrdquo BehaviouralBrain Research vol 208 no 2 pp 643ndash645 2010