Caspase inhibitor infusion protects an avian song control circuit from seasonal-like neurodegeneration.

Abstract

Sex steroids such as androgens and estrogens have trophic effects on the brain and can ameliorate neurodegeneration, and the withdrawal of circulating steroids induces neurodegeneration in several hormone-sensitive brain areas. Very little is known about the underlying molecular mechanisms that mediate neuronal regression caused by hormone-withdrawal, however. Here we show that reduction of programmed cell death by local infusion of caspase inhibitors rescues a telencephalic nucleus in the adult avian song control system from neurodegeneration that is induced by hormone withdrawal. This treatment also has trans-synaptic effects that provide some protection of an efferent target region. We found that unilateral infusion of caspase inhibitors in vivo in adult white-crowned sparrows rescued neurons within the hormone-sensitive song nucleus HVC (used as a proper name) from programmed cell death for as long as seven days after withdrawal of testosterone and a shift to short-day photoperiod and that the activation of caspase-3 was reduced by 59% on average in the ipsilateral HVC compared with the unmanipulated contralateral HVC. Caspase inhibitor infusion near HVC was sufficient to preserve neuron size ipsilaterally in a downstream nucleus, the robust nucleus of the arcopallium. This is the first report that sustained local application of caspase inhibitors can protect a telencephalic brain area from neurodegeneration in vivo and that a degenerating neural circuit rescued with caspase inhibitors produces sufficient trophic support to protect attributes of a downstream target that would otherwise degenerate. These results strengthen the case for the possible therapeutic use of caspase inhibitors under certain neurodegenerative conditions.

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Behavioral/Systems/Cognitive
CaspaseInhibitorInfusionProtectsanAvianSongControl
CircuitfromSeasonal-LikeNeurodegeneration
ChristopherK.Thompson1andEliotA.Brenowitz1,2
1GraduatePrograminNeurobiologyandBehaviorand2DepartmentsofPsychologyandBiology,UniversityofWashington,Seattle,Washington98195-
1525
Sex steroids such as androgens and estrogens have trophic effects on the brain and can ameliorate neurodegeneration, and the with-
drawal of circulating steroids induces neurodegeneration in several hormone-sensitive brain areas. Very little is known about the
underlying molecular mechanisms that mediate neuronal regression caused by hormone-withdrawal, however. Here we show that
reductionofprogrammedcelldeathbylocalinfusionofcaspaseinhibitorsrescuesatelencephalicnucleusintheadultaviansongcontrol
systemfromneurodegenerationthatisinducedbyhormonewithdrawal.Thistreatmentalsohastrans-synapticeffectsthatprovidesome
protectionofanefferenttargetregion.Wefoundthatunilateralinfusionofcaspaseinhibitorsinvivoinadultwhite-crownedsparrows
rescued neurons within the hormone-sensitive song nucleus HVC (used as a proper name) from programmed cell death for as long as
sevendaysafterwithdrawaloftestosteroneandashifttoshort-dayphotoperiodandthattheactivationofcaspase-3wasreducedby59%
on average in the ipsilateral HVC compared with the unmanipulated contralateral HVC. Caspase inhibitor infusion near HVC was
sufficienttopreserveneuronsizeipsilaterallyinadownstreamnucleus,therobustnucleusofthearcopallium.Thisisthefirstreportthat
sustained local application of caspase inhibitors can protect a telencephalic brain area from neurodegeneration in vivo and that a
degenerating neural circuit rescued with caspase inhibitors produces sufficient trophic support to protect attributes of a downstream
target that would otherwise degenerate. These results strengthen the case for the possible therapeutic use of caspase inhibitors under
certainneurodegenerativeconditions.
Keywords:apoptosis;birdsong;caspase;neuroendocrine;neuroethology;neuroprotection;plasticity;testosterone
Introduction
Sex steroids such as testosterone (T) provide trophic support to
hormone-sensitive brain structures in adult animals. The sup-
portive role played by T is best demonstrated by withdrawal of
hormones after castration, which leads to regression of various
neuronalattributesinseveralbrainareasacrossseveralvertebrate
taxa (Breedlove and Arnold, 1981; Commins and Yahr, 1984;
Panzicaetal.,1987;Wadeetal.,1993;Ielaetal.,1994;Somaetal.,
1996; Cooke et al., 1999). Furthermore, T and its metabolites
17-?estradiol(E2)and5-?dihydrotestosterone(DHT)canpro-
tect neurons under various neurodegenerative conditions
(Garcia-Segura et al., 2001; Veiga et al., 2004). Indeed, de-
clines of circulating levels of T in aging men are implicated in
accelerating cognitive decline and exacerbating neurodegen-
erative disorders (Veiga et al., 2004). However, despite the
occurrence of this phenomenon across taxa and its clinical
relevance to aging men, little is known about the underlying
molecular mechanisms that mediate neurodegeneration
caused by the withdrawal of circulating T.
Song behavior in oscine passerines is regulated by a series of
discrete brain nuclei known as the song control system (see Fig.
1A). This brain circuit serves as an excellent model for under-
standing hormone-dependent neurodegeneration because many
nuclei normally undergo substantial seasonal changes that are
related to the reproductive status of the animal, especially circu-
lating levels of T. Seasonal-like regression in the song control
system stands out among other models of hormone-mediated
brain plasticity because these changes are rapid and robust
(Thompson et al., 2007). When circulating T is rapidly with-
drawnfrombreeding-conditionadultmalewhite-crownedspar-
rows(Zonotrichialeucophrys),HVC(usedasapropername)vol-
ume regresses quickly, initially driven by an increase in neuronal
density within 12 h, followed by a 26% decrease in neuron num-
ber2–4dlater.Thevolumesofrobustnucleusofthearcopallium
(RA) and Area X, efferent nuclei of HVC, regress more slowly
overdaystoweeksandresultprimarilyfromchangesinneuronal
size and density but not neuron number (Thompson and Bre-
nowitz, 2005; Thompson et al., 2007).
Seasonal growth of RA and Area X is dependent on trans-
synaptic afferent input from HVC. Unilateral lesion of HVC
blocks the growth of ipsilateral RA and Area X in response to
systemicadministrationofTandexposuretolong-dayphotope-
riod (LD) (Brenowitz and Lent, 2001). In addition, an intracere-
bral T implant placed near HVC in nonbreeding male white-
crownedsparrowsinducesthegrowthoftheipsilateralHVC,RA,
ReceivedFeb.13,2008;revisedMay19,2008;acceptedMay28,2008.
This work was supported by National Institutes of Health Grants MH53032, P30DC04661, DC03829, T32-
DC05361,and5T32-GM07108.WethankE.Rubel,K.Lent,andP.Berberianfortechnicalassistance.
CorrespondenceshouldbeaddressedtoChristopherK.Thompson,UniversityofWashington,Box351525,Seat-
tle,WA98195-1525.E-mail:ckthomps@u.washington.edu.
DOI:10.1523/JNEUROSCI.0663-08.2008
Copyright©2008SocietyforNeuroscience 0270-6474/08/287130-07$15.00/0
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and Area X (Brenowitz and Lent, 2002).
This transsynaptic influence is driven by
theactionofTinHVC;aTimplantplaced
near RA does not promote the growth of
ipsilateral song control nuclei. Infusion of
DHT and E2near HVC in male birds un-
der short-day photoperiod (SD) is suffi-
cient to induce growth of ipsilateral RA
soma area; infusion near RA has no effect
(Meitzen et al., 2007).
Although little is known about the mo-
lecular mechanisms that underlie neuro-
nalregressioncausedbyTwithdrawal,de-
generation of brain circuits typically
involvestheactivationofprogrammedcell
death pathways (Mattson, 2000; Okouchi
et al., 2007). In particular, caspases are a
family of proteases that play an integral
roleinprogrammedcelldeathpathwaysin
vertebrates and invertebrates (Kumar,
2006). The use of inhibitors that reduce
the activity of various caspases have eluci-
dated the role of caspase-dependent pro-
grammed cell death and may have clinical
significance (Ray, 2006). Here we block
caspaseactivationbyinfusingamixtureof
caspaseinhibitorsnearHVCononesideof
the brain in vivo to protect the ipsilateral
song controlsystem
degeneration.
circuitfrom
MaterialsandMethods
All procedures followed National Institutes of Health animal use guide-
lines and were approved by the University of Washington Institutional
AnimalCareandUseCommittee.Wecaptured15maleGambel’swhite-
crowned sparrows (Zonotrichia leucophrys Gambelii) in eastern Wash-
ingtonduringtheirpostbreedingseasonmigration.Wehousedthebirds
inindoorgroupaviariesforatleast12weeksonSD(8hlight)beforethe
start of the experiment to ensure that the song system and reproductive
system were fully regressed and sensitive to the stimulatory effects of T
and long-day photoperiods.
Thesongsystemnucleiinwildmalewhite-crownedsparrowsincrease
in size in response to a gradual increase in circulating T levels as day
length increases and the testes grow. The timing of the increase in circu-
lating T levels varies across individuals, however. To reduce individual
variability, we exposed all birds to the same long-day photoperiod and
administered T subcutaneously to rapidly elevate plasma T concentra-
tions to those seen in breeding birds (4–12 ng/ml) (Wingfield and
Farner,1978).Althoughthistransitionoccursmoregraduallyinthewild,
this laboratory manipulation recreates the two most important seasonal
influences on white-crowned sparrows: elevated T levels and a long-day
photoperiod typical of their Alaskan breeding grounds (20 h of light per
day).Itofferstheadvantageofprovidingadiscretestartingpointfortime
course studies of growth and regression of song circuits.
At the beginning of the experiment, we transferred the birds from an
8/16 h light/dark cycle (SD) to 20/4 h light/dark cycle (LD) overnight
(Fig. 1B). The next day, we anesthetized each bird with isoflurane
through a non-rebreathing system and castrated them. We made a small
incision on the left side anterior to the caudalmost rib and dorsal to the
uncinateprocessandaspiratedbothtestes.Oncecastrated,weimplanted
each bird subcutaneously with a 12 mm SILASTIC capsule (inner diam-
eter ? outer diameter, 1.47 ? 1.96 mm) filled with crystalline T (Ster-
aloids).WecastratedthebirdsattheonsetofLDtoavoidsubjectingbirds
to this additional surgery when they were later implanted with intrace-
rebral cannulas at the transition from breeding to nonbreeding condi-
tions. We housed the birds individually in visual and auditory contact
with the other birds used in this experiment. We kept all the birds on
long-dayphotoperiodandhighlevelsofcirculatingT(LD?T)for28d.
This time period is sufficient to induce full growth of the song control
system under these conditions (Tramontin et al., 2000).
After 26 d of LD ? T conditions, we implanted a cannula attached to
an osmotic pump unilaterally near HVC in each bird. We anesthetized
eachbirdwithisofluraneandplaceditintoastereotaxicheadholder.We
made an incision into the scalp, removed the portion of the skull overly-
ing the midsagittal sinus, and used the bifurcation of the midsagittal
sinus as the zero point. We randomly chose a hemisphere and lowered
the cannula 0.75 mm into the telencephalon just caudal to HVC (lateral,
2.8mm;posterior,0.4mm).Wefixedthecannulatotheskullwithdental
cement and attached an osmotic pump (Alzet) to the cannula. Twelve
birds received osmotic pumps filled with a mixture of caspase inhibitors
(0.015 mg in 100 ?l in 1% DMSO in equal concentrations: pan-caspase
inhibitor (Z-VAD-FMK; R&D Systems), caspase-3 inhibitor (Z-DEVD-
FMK; Calbiochem), and caspase-9 inhibitor (Z-LEHD-FMK; Calbio-
chem).ThreebirdsreceivedosmoticpumpsfilledwithZ-FA-FMK(Cal-
biochem),anegativecontrolforcaspaseinhibitors(0.015mgin100?lin
1% DMSO) (Ekdahl et al., 2001). We placed the osmotic pumps into
microcentrifuge tubes filled with avian saline (0.9% NaCl), sealed the
tubes with paraffin and quick-drying cement, and mounted the tubes
into“backpacks”custommadeforwhitecrownedsparrows.Theosmotic
pump rested between the bird’s wings and allowed for free movement.
Osmoticpumpsreleasedtheircontentssimilarlytothoseimplantedsub-
cutaneously, and the backpack system reduced the risks of secondary
infection, extrusion of pumps, and mortality that can occur with subcu-
taneous implant.
WeremovedthesubcutaneousTcapsule2dafterthecannulaimplan-
tation to ensure the onset of infusion of caspase inhibitors (or negative
control) into HVC before T withdrawal. We shifted the birds overnight
toa14/10hlight/darkcyclethesamedayastheTwithdrawalandthenext
day shifted them overnight to SD. The intermediate photoperiod helped
birds adjust to the reduction in available feeding time. We killed groups
Figure1.
controlsystemshowingprojectionsofmajornuclei.Thegreenarrowsindicatethedescendingmotorpathway.Theredarrows
indicate the anterior forebrain pathway. The blue arrow indicates auditory input into the song control system, which includes
caudalmesopallium,caudalnidopallium,andnucleusinterfacialis.B,Schematictimelineoftheexperimentalprocedures.Groups
ofanimalsthatreceivedunilateralinfusionofcaspaseinhibitorswerekilledondays1,3,and7.Animalsthatreceivedunilateral
infusion of negative control (Neg. Cont.) for caspase inhibitors were killed on day 7. DLM, Medial nucleus of the dorsolateral
thalamus;nXIIts,tracheosyringealportionofthehypoglossalmotornucleus;lMAN,lateralportionofthemagnocellularnucleusof
theanteriornidopallium;X,AreaX;Sac,sacrifice.
Theaviansongcontrolsystemandtimelineforexperimentalprocedures.A,Schematicsagittaldiagramofthesong
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of birds 1 d (n ? 3), 3 d (n ? 4), and 7 d (caspase inhibitor mixture, n ?
5; negative control, n ? 3) after T withdrawal, which we consider as the
onset of nonbreeding conditions.
Blood draw and hormone analysis. We took blood samples from the
birds at various time points throughout the experiment to measure cir-
culating T levels. We withdrew 250 ?l of blood from the alar vein in the
wing into heparinized collection tubes. We immediately centrifuged the
tubes to separate the plasma, which was stored at ?20°C until assay. We
measured plasma T concentration using the Coat-A-Count Total Tests-
terone radioimmunoassay kit (Diagnostic Products). The minimum de-
tectableplasmaTconcentrationwas0.09ng/ml.Sampleswithundetect-
able levels of steroid were treated as having concentrations at this
detection limit for statistical analysis.
Perfusion and tissue processing. We deeply anesthetized the birds with
isofluraneinhalationandperfusedthemthroughtheheartwithheparin-
ized saline, followed by 4% phosphate-buffered paraformaldehyde (pH
7.4). We postfixed the brains in 4% paraformaldehyde for 24 h, embed-
ded the brains in gelatin, and postfixed the gelatin-embedded brains in a
20% sucrose–neutral buffered Formalin solution for 48 h. We sectioned
the brains in the coronal plane at 40 ?m on a freezing microtome,
mountedeverythirdsection,andstainedwiththionin.Wekeptalternate
sections in saline (0.7%) for immunohistochemistry (see below).
Measurementofnucleivolume.Usinganoverheadprojector,wetraced
the borders in both hemispheres throughout the full rostrocaudal extent
of HVC, RA, and Area X. We scanned these tracings into a microcom-
puter and measured the surface area of each cross section using a cus-
tomizedmoduleofNIHImage.Wedeterminedthevolumesofnucleiby
summing the estimated volume between sections calculated with the
formula for the volume of a cone frustum (Tramontin et al., 1998).
Measurement of neuronal attributes in HVC and RA. We sampled neu-
ron size by measuring the cross-sectional area of the soma in every
mounted section throughout the rostrocaudal axis of HVC and RA. We
distinguished neurons from glia by their single nucleolus and uniform,
nongranular cytoplasm. We used a random, systematic sampling proto-
col that has been previously described and validated (Tramontin et al.,
1998), which yields estimates of neuronal density and size that do not
differ from those obtained using the stereological optical dissector
method.Wemeasuredthesomaticareaofatleast150HVCneuronsand
100 RA neurons per hemisphere of each bird in fields chosen randomly
by computer in each section. In thin sections, there is a likelihood of
splitting of neuronal nuclei between sections, which could overestimate
cell counts (West, 1993; Coggeshall and Lekan, 1996). To estimate neu-
ron density, we therefore counted neuronal nucleoli in every field and
used Konigsmark’s (Nauta and Ebbesson, 1970) formula 4 to correct for
nucleus splitting: N/n ? t/(t ? 2(sqrt(r2? (k/2)2)), where N is the
number of nucleoli present, n is the number of nucleoli counted, t is the
section thickness in micrometers, r is the nucleolus radius, and k is the
diameteroftheuncountedfragmentsofnucleoli.Wesetkequalto1?m,
whichequaledthesmallestnucleolusfragmentencounteredinthisstudy.
Konigsmark-correctedneuroncountsweredividedbythevolumeofthe
tissue sampled to obtain neuronal density.
We estimated neuron number by multiplying neuron density by total
nucleus volume. We sampled at least 150 HVC neurons and 100 RA
neurons throughout the rostrocaudal extent of each nucleus in each
brain. This sample size is sufficient to encompass the entire range of
variability in neuron density and somatic area in these nuclei, based on
Tramontin et al. (1998). All measurements were made blind to the time
of death for each bird.
Caspase-3 immunohistochemistry. To determine whether caspase in-
hibitor infusion sufficiently decreased caspase activation, we performed
immunohistochemistry for activated caspase-3, a protease that mediates
programmed cell death. We rinsed floating sections containing con-
tralateral and ipsilateral HVC in three washes of 0.1 M PBS with 0.1%
Triton X-100 (PBS-TX), pH 7.4, and transferred them to 90–95°C, 10
mM sodium citrate (pH 6.0) for 20 min. We allowed the sections to cool
to room temperature for 20 min, rinsed them in PBS-TX three times,
preblocked the tissue in 10% normal goat serum for 1 h, and incubated
thetissuewithaprimaryantibodyagainsttheactivatedformofcaspase-3
(1:1000 in 10% NGS; Abcam) overnight at 4°C. We washed the sections
inthreewashesofPBS-TX,blockedendogenousperoxidaseactivitywith
10minof3%H2O2in10%MeOHand0.1 MPBS,washedthesectionsin
three washes of PBS-TX, and incubated the sections for 45 min in 0.01 M
PBS containing the secondary antibody (1:200). We visualized with avi-
din–biotinamplification(VectastainElite;VectorLaboratories)anddia-
minobenzidine chromagen. Negative control tissue was prepared by
omitting either the primary or the secondary antibody. These sections
showed no staining of cells or neuropil. We mounted the sections onto
gelatin-subbed slides, let them air dry overnight, and coverslipped the
slides with DPX mountant.
To quantify the activation of caspase-3, we scanned the entire cross-
sectionalareaofHVCandcountedallcellswithintheborderofHVCthat
showedpositivecytosoliclabelingthatwashigherthanbackground.The
observer was blind to treatment.
Statistics.Weusedatwo-tailedpairedttesttocompareipsilateralwith
contralateral attributes, a power analysis on paired t tests to assess the
likelihood of false negatives, and two-way ANOVAs and t tests to com-
pare the regression of contralateral HVC attributes with previously pub-
lished data (Thompson et al., 2007). The ? level for all tests was 0.05.
Results
We measured the volume of song nuclei from Nissl-stained sec-
tions.GiventhattheprojectionfromHVCtoRAisunilateraland
bilaterally symmetrical, unilateral infusion of caspase inhibitors
leaves the untreated contralateral hemisphere to serve as a
within-animal control. We therefore compared measurements
ipsilateral to the infusion with the contralateral hemisphere with
paired t tests (? ? 0.05, two-tailed). Caspase inhibitor infusion
somewhat reduced the regression of HVC volume 1 d after T
withdrawal and photoshift, but this reduction was not statisti-
cally significant ( p ? 0.100). We found that infusion of caspase
inhibitors did, however, significantly rescue ipsilateral HVC vol-
ume3d( p?0.039)and7d( p?0.003)afterTwithdrawaland
photoshift(Fig.2A).Infusionofacaspaseinhibitornegativecon-
trol for 7 d did not preserve HVC volume ( p ? 0.321) (Fig. 2A).
We measured HVC neuronal attributes from Nissl-stained
sections using a random, systematic counting scheme. Caspase
inhibitor infusion significantly preserved the area of the soma of
HVCneurons7d( p?0.021)afterTwithdrawalandphotoshift
(Fig. 2B). Caspase inhibitor infusion somewhat preserved HVC
soma area 1 and 3 d after T withdrawal and photoshift, but this
reduction was not statistically significant ( p ? 0.078 and 0.090,
respectively) (Fig. 2B). The regression of HVC volume attribut-
abletothewithdrawalofTandshifttoSDisinitiallymediatedby
an increase in neuron density over the first few days, followed by
a subsequent decrease in neuron number (Thompson et al.,
2007). Consistent with these observations, infusion reduced the
increase in HVC neuron density at 1 d ( p ? 0.018) and 3 d ( p ?
0.033)(Fig.2C).At7d,neitherinfusionofcaspaseinhibitorsnor
negative control had any effect on HVC neuron density ( p ?
0.511 and 0.879, respectively) (Fig. 2C). Caspase inhibitor infu-
sion protected HVC neurons from loss 3 d ( p ? 0.040) and 7 d
( p?0.005)buthadnoeffect1d( p?0.325)aftermanipulation
(Fig. 2D). Infusion of negative control did not prevent HVC
neuron loss 7 d after manipulation ( p ? 0.081).
CaspaseinhibitorinfusionnearHVCprotectedsome,butnot
all, neuronal attributes of ipsilateral RA, an efferent target of
HVC. Infusion of caspase inhibitors significantly preserved RA
soma area 7 d ( p ? 0.030) but not 1 d ( p ? 0.464) and 3 d ( p ?
0.499) after T withdrawal and photoshift (Fig. 3). Infusion of
caspase inhibitor negative control did not prevent the regression
of RA soma area ( p ? 0.597). Infusion of caspase inhibitors had
noeffectonnucleusvolumeofRA,RAneurondensity,orneuron
number at any time point examined (supplemental Fig. 1, avail-
able at www.jneurosci.org as supplemental material).
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We measured the volume of Area X, an
efferenttargetofHVCneurons,andfound
that it did not differ across hemispheres in
anytimepoint(supplementalFig.2,avail-
ableatwww.jneurosci.orgassupplemental
material).
Caspase inhibitor infusion reduced the
incidence of cells positive for activated
caspase-3 3 d after T withdrawal and pho-
toshift (Fig. 4A). We found that caspase
inhibitor infusion reduced the density
( p ? 0.010) and number ( p ? 0.040) of
activatedcaspase-3-positivecellsintheip-
silateral HVC (Fig. 4B,C). In addition,
caspase inhibitor infusion reduced the
number of activated caspase-3-positive
cells per 1000 HVC neurons ( p ? 0.012,
data not shown). We processed tissue
from 1 d animals for activated caspase-3
and found relatively little labeling; histol-
ogy from one bird did not allow us to an-
alyze the data quantitatively. Also, we had
not yet optimized immunostaining for ac-
tivated capase-3 when we processed tissue
from 7 d animals.
We performed power analysis on some
marginallysignificantresultstodetermine
the likelihood that the outcome may be
explained by insufficient power. There
werethreeresultsthatweremarginallysig-
nificant(0.05?pvalue?0.1)andshowed
differences across hemispheres ?10%; we
use this level as a criterion for assessing
whether a difference across hemispheres
may be meaningful. Soma area was 12.9
and 15.0% larger in the ipsilateral HVC in
the1and3dayanimals,respectively.ThepvaluesforHVCsoma
area in the 1 and 3 d animals were 0.078 and 0.09 with power of
0.422 and 0.345, however. Ipsilateral HVC volume was 23%
larger than the contralateral HVC in the 1 d animals, and the p
valuewas0.10withpowerof0.34.Thesemarginalresultsmaybe
attributabletoseveralfactors,includingrelativelylowNandhigh
variability across animals. These marginal results are not solely
attributable to low N, however. HVC neuron density was signif-
icantlydifferentacrosshemispheresinthe1dgroup( p?0.018),
and the power was 0.933. This shows that statistical differences
with sufficiently high power can be obtained from paired t tests
withonlythreeanimals.Nevertheless,thethreeresultswithmar-
ginal results discussed above should be interpreted cautiously.
We performed two types of statistical analysis to determine
whether the regression in the contralateral HVC that we report
hereiscomparablewithpreviouslypublishedresults(Thompson
et al., 2007): two-way ANOVAs comparing the current dataset
with those from Thompson et al. (2007) using two time points
from both studies, and t tests comparing LD ? T data from
Thompson et al. (2007) with 7 d data from the current study. In
summary,resultsfromtwo-wayANOVAsandttestsshowedthat
contralateral neuronal attributes in the current study regressed
following a similar time course to that reported in Thompson et
al. (2007). Details of these analyses can be found in the supple-
mental materials.
We measured circulating levels of T of each bird 7–14 d after
the initiation of LD ? T and again when the animals were killed.
CirculatingTlevelsforallbirdsusedinthestudywhileunderLD
conditions(12.78?1.03ng/ml;mean?SEM)werecomparable
withthoseseeninbreedingGambel’swhite-crownedsparrowsin
the wild (Wingfield and Farner, 1978). Circulating levels of T
when the animals were killed (0.62 ? 0.15 ng/ml) were below 1
ng/ml, which, together with visual verification of the absence of
testes at the time of death, confirmed successful withdrawal of
circulating T and transition to nonbreeding hormonal state. The
intra-assay coefficient of variation was ?5%, and the interassay
coefficient of variation was 12%. We did not collect enough
plasma from one animal when the animals was killed and had to
exclude it from hormone analysis. Visual inspection after the
animals were killed confirmed that this animal had no testes,
however, and was therefore included in the study.
Discussion
Our results show that the rapid regression of HVC caused by T
withdrawal and photoshift is dependent on the activity of
caspases. The infusion of a mixture of caspase inhibitors near
HVC in vivo results in the near-complete preservation of ipsilat-
eral HVC after the withdrawal of circulating T and transition to
SD. The volume of HVC normally regresses within 12 h after the
withdrawal of T; continuous infusion of caspase inhibitors pre-
vents this regression for nearly 7 d. Conversely, infusion of a
negative control for caspase inhibitors did not preserve the ipsi-
lateral HVC, which demonstrates that the protective effects of
caspaseinhibitorinfusioninHVCareattributabletotheactionof
Figure 2.
conditions.Eachanimalwithineachgroupisrepresentedbyadistinctcoloracrossallfigures.Circlesrepresentmeasurements
takeninipsilateralHVC,andsquaresrepresentmeasurementstakenincontralateralHVC.*p?0.05,**p?0.01,***p?0.005,
significantdifferencesacrosshemispheres(pairwisettest).A,CaspaseinhibitorssignificantlyprotectedipsilateralHVCvolume3
and7daftertransitiontononbreedingconditions.B,CaspaseinhibitorssignificantlypreservedipsilateralHVCsomaarea7dafter
transition.C,CaspaseinhibitorssignificantlyreducedtheincreaseinipsilateralHVCneurondensity1and3daftertransition.D,
CaspaseinhibitorssignificantlyprotectedipsilateralHVCfromneuronloss3and7daftertransition.Infusionofanegativecontrol
for caspase inhibitors (Neg. Cont.) did not prevent regression of any attribute of HVC 7 d after the transition to nonbreeding
conditions.
In vivo infusion of caspase inhibitors preserved ipsilateral HVC after the transition from breeding to nonbreeding
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caspaseinhibitorsandnotattributabletosomenonspecificeffect
ofthevehicleorthesurgicalimplantationofthecannula.Caspase
inhibitors had protective effects on every neuronal attribute of
HVC that we measured, including a significant increase in the
survival of HVC neurons. In addition, we showed that there was
a high number of activated caspase-3-positive cells in the con-
tralateralHVC3dafterthetransitiontononbreedingconditions
andthatinvivoinfusionofcaspaseinhibitorsreducedtheactiva-
tionofcaspase-3inipsilateralHVC.Together,theseresultsshow
thatactivationofcaspasesisnecessaryfortheseasonalregression
of HVC.
The withdrawal of circulating T from adult male white-
crowned sparrows induces a rapid regression of HVC volume to
nonbreeding size within 12 h, and this initial decline is attribut-
abletoasubstantialincreaseinneurondensity(Thompsonetal.,
2007). This increase in neuron density reflects a decrease in ex-
tracellular space and strongly suggests the occurrence of signifi-
cantatrophyofaxonaland/ordendriticarborswithintheneuro-
pil of HVC. There is precedent for caspase-dependent
modification of dendritic and axonal arbors; for instance, the
activation of caspases is necessary for the pruning of dendrites of
sensory neurons during the metamorphosis of Drosophila mela-
nogaster (Williams et al., 2006). The activation of caspases can
regulatesynapticplasticitywithinneuronswithouttheactivation
of programmed cell death pathways that lead to apoptosis. In
zebra finch auditory forebrain, there is a brief increase of
caspase-3 activation in dendritic spines within minutes of expo-
sure to novel song with no subsequent neuron loss (Huesmann
and Clayton, 2006). Our results suggest that increases in HVC
neuron density, if driven by degenerating neuropil, may be
caspasedependentgiventhatinvivoinfusionofcaspaseinhibitor
preventedtheincreaseinneurondensity1and3daftertransition
to nonbreeding conditions. Our data, however, do not necessar-
ily indicate that caspase inhibitors solely affect dendritic and/or
axonal pruning, despite the fact that caspase inhibitors preserve
ipsilateral HVC neuron density. Instead, the initial increase in
HVC neuron density after the transition to nonbreeding condi-
tions may be a result of the retraction of dendrites and axons of
HVC neurons that have already initiated programmed cell death
andeventuallydiedayslater,whereasthearborsofHVCneurons
thatsurvivemaynotundergodegeneration.Inaddition,changes
inHVCneurondensitymaybetheresultofchangeswithinnon-
neuronalcellssuchasgliaorendothelialtissue,bothofwhichare
sensitive to changes in circulating levels of T (Louissaint et al.,
2002; Satriotomo et al., 2004). Future studies that specifically
examine the underlying mechanisms that mediate rapid degen-
eration of HVC neuropil, including dendritic and/or axonal
pruning or regression of non-neuronal cells within the hours
after T withdrawal, may reconcile this issue.
NeuronnumberinHVCdecreasesby26%within7dafterthe
transitiontononbreedingconditions(Thompsonetal.,2007).In
vivo infusion of caspase inhibitors, however, provides complete
protection of HVC neurons under these conditions; on average,
ipsilateral HVC neuron number was 28% greater than contralat-
eral HVC at 7 d. Caspase inhibitors also preserved HVC soma
Figure3.
ipsilateralRAneurons7dafterthetransitiontononbreedingconditions.Eachanimalwithin
eachgroupisrepresentedbyadistinctcolor.Circlesrepresentmeasurementstakeninipsilateral
RA; squares represent measurements taken in contralateral RA.*p ? 0.05, significant differ-
enceacrosshemispheres(pairwisettest).
InvivoinfusionofcaspaseinhibitorsnearHVCsignificantlypreservedsomaareaof
Figure4.
ofcapsase-3inipsilateralHVC3dafterthetransitionfrombreedingtononbreedingconditions.
A,Examplesofimmunohistochemistryforactivatedcaspase-3inonesectiontakenfroma3d
bird.B,Caspaseinhibitorssignificantlyreducedthedensityofactivatedcaspase-3-positivecells
inipsilateralHVC.C,Caspaseinhibitorssignificantlyreducedthenumberofactivatedcaspase-
3-positive cells in ipsilateral HVC. Each animal is represented by a distinct color. *p ? 0.05,
**p?0.01,significantdifferencesacrosshemispheres(pairwisettest).Scalebars,100?m.
InvivoinfusionofcaspaseinhibitorsnearHVCsignificantlyreducedtheactivation
7134 • J.Neurosci.,July9,2008 • 28(28):7130–7136ThompsonandBrenowitz•CaspaseInhibitorsPreventSongSystemfromRegression

Page 6

area for at least 7 d after T withdrawal and photoshift; average
ipsilateral HVC soma area was 13% larger than contralateral,
comparablewithmeasurementsseeninbirdskilledunderbreed-
ing conditions (Thompson et al., 2007). In addition, two-way
ANOVAs and t tests show that the regression of the contralateral
HVCwascomparablewithpreviouslypublishedresults(Thomp-
son et al., 2007). These results strongly suggest that infusion of
caspase inhibitor provides full protection for several neuronal
attributes that otherwise are compromised under seasonal-like
transition to nonbreeding conditions.
Although our results are the first demonstration that in vivo
administrationofcaspaseinhibitorscanameliorateneurodegen-
eration attributable to the withdrawal of circulating sex steroids,
caspase inhibitors have been used successfully to rescue brain
areas affected by neurodegeneration attributable to other causes.
Ischemia and status epilepticus induce programmed cell death
andsubsequentneuronlossinthemammalianhippocampusand
cortex, and in vivo administration of caspase inhibitors reduces
this process (Himi et al., 1998; Mattson et al., 2000; Ekdahl et al.,
2002; Reshef et al., 2007). Caspase inhibitors also increase the
short-term survival of new neurons incorporated into the hip-
pocampus (Ekdahl et al., 2001). Indeed, the balance of incorpo-
ration and death of neurons in the dentate gyrus can be affected
by acute injections of caspase inhibitors in rats (Dupret et al.,
2007). Caspase inhibitors have been shown to influence of the
consolidationofnewmemoriesinmammals(Dupretetal.,2007)
and birds (Huesmann and Clayton, 2006). Given these effects,
caspase inhibitors may prove to have clinical application in ame-
liorating neurodegenerative processes in humans (Ray, 2006).
Although it has not yet been conclusively shown in white-
crowned sparrows, studies in zebra finches suggest that the only
HVC neurons that are replaced in adults are those that project to
RA (Scotto-Lomassese et al., 2007). Neurons rescued with
caspaseinhibitorsinsparrowsafterthetransitiontononbreeding
conditions may also be mostly neurons that project to RA. Our
results show that in vivo infusion of caspase inhibitors near HVC
reduced the regression of average soma area in ipsilateral RA 7 d
after the transition to nonbreeding conditions. The observation
of a trans-synaptic trophic influence of HVC on RA neurons is
consistent with previous results. Lesions of HVC block the
growth and maintenance of RA in sparrows exposed to breeding
conditions(BrenowitzandLent,2001),demonstratingthatsome
characteristic of HVC, possibly increased release of trophic fac-
tors and/or enhanced electrophysiological activity of RA-
projectingHVCneurons,isnecessaryforthegrowthofRA(A.M.
Wissman and E. A. Brenowitz, unpublished results) (Meitzen et
al., 2007). This transsynaptic rescue of RA is not complete, how-
ever; infusion of caspase inhibitors near HVC preserved ipsilat-
eral RA soma area but had no effect on RA neuron density. If
caspase inhibitors were to have an effect on RA neuron density,
we would expect to see it here because density is significantly
increased by 2 d after the transition to nonbreeding conditions
and continues to increase thereafter (Thompson et al., 2007).
There are several possible explanations for these observations.
First, we only used one dose in our study; preserving RA neuron
density, which likely reflects changes in dendritic arborization,
mayrequireahigherconcentrationofcaspaseinhibitors.Second,
RAsomaareaandneurondensitymaybedifferentiallyregulated,
andcaspaseinhibitorsmaynotbesufficienttopreventchangesin
dendritic arborization and/or axonal terminal fields that likely
determine RA neuron density. In addition, there is precedent for
dissociation of dendritic changes and soma size; Deitch and
Rubel (1984) found that deafferentation of nucleus laminaris
neurons induced dendritic retraction with no change in soma
size,furthersuggestingthatsomaareaanddendriticarborization
may be differentially regulated. Last, the rapid transition to non-
breeding conditions initiates a cascade of events that leads to the
activation of caspases and ultimately neurodegeneration. The
pointatwhichcaspaseinhibitorsexerttheirinfluenceisrelatively
faralongthecausalchainofeventsthatmediateprogrammedcell
death (Danial and Korsmeyer, 2004). Thus, caspase inhibitors
may not provide the full complement of support for compro-
mised HVC neurons that are necessary for transsynaptic influ-
ence over RA neurons. Nevertheless, our results suggest that a
population of HVC neurons that would otherwise be eliminated
via programmed cell death pathways in the days after the transi-
tion to nonbreeding conditions are rescued with caspase inhibi-
tors and maintain enough integrity to have some transsynaptic
influence on the morphology of efferent RA neurons.
Although photoperiod and song rate have some influence
over morphological change within the song control system
(Smith et al., 1997; Bentley et al., 1999; Sartor and Ball, 2005), T
and/or its metabolites play a much more direct and significant
role (Smith et al., 1997). As noted above, T acts locally within
HVC to induce the growth of ipsilateral song control system
nuclei (Brenowitz and Lent, 2002), including a significant in-
creaseinHVCneuronnumber.Thus,itseemsthat,underbreed-
ing conditions, elevated levels of circulating T promotes the sur-
vival of HVC neurons, which in turn induces the growth of
efferentnuclei,andthattherapidwithdrawalofTinitiatesarapid
caspase-dependent degeneration of the song control system.
TheseobservationsstronglysuggestthatTand/oritsmetabolites
play a neuroprotective role in HVC not unlike the hormone-
mediated neuroprotection that is seen in several in vivo animal
models of neuronal injury (Ramsden et al., 2003; Pike et al.,
2006). In mammals, T and E2upregulate the expression of anti-
apoptoticgenes(Singeretal.,1998;Dubaletal.,1999;Pike,1999;
Stoltzner et al., 2001; Zup and Forger, 2002; Chiueh et al., 2003;
Wuetal.,2005).Inzebrafinches,neuronalinjuryisaccompanied
by an increase in expression of aromatase, an enzyme that con-
verts T to E2(Peterson et al., 2001), and inhibition of aromatase
increases apoptosis near the injury (Saldanha et al., 2005).
Seasonal-likerapidregressionofthesongcontrolsystemservesas
an excellent model to further elucidate the molecular mecha-
nisms that underlie hormone-mediated neurodegeneration.
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