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Research Report

Neurocalcin protein labeling reveals a dimorphism within thedeveloping zebra finch brain

Philip Long, Sean L. Veney⁎

Department of Biological Sciences, Kent State University, Kent, OH 44242, USA

A R T I C L E I N F O

⁎ Corresponding author. Fax: 330 672 3713.E-mail address: sveney@kent.edu (S.L. Ve

0006-8993/$ – see front matter © 2009 Publisdoi:10.1016/j.brainres.2009.11.083

A B S T R A C T

Article history:Accepted 30 November 2009Available online 8 January 2010

The exact mechanism(s) responsible for sexual differentiation of the zebra finch songsystem remains unknown but likely involve a combination of hormonal and genetic factors.One product that may play a role is the calcium binding protein, neurocalcin. A previousstudy indicated that neurocalcin mRNA was widely distributed throughout the zebra finchtelencephalon, overlapping with song control regions. Because it was not clear howmuch ofthat expression was functionally relevant, we labeled tissue with a specific antibody. At thethree age ranges that were examined, there were region-specific fluctuations in the numberof neurocalcin immunoreactive cells. During post-hatching developmental ages (P) 18–23,quantitative analyses indicated that females had significantly more neurons containingneurocalcin protein in HVC and RA thanmales. This difference was not detected at P10 or inadults. Labeling in LMAN did not reveal a sex difference at any of the ages. In other areas ofthe brain, semi-quantitative analyses of the relative number of cells immunopositive forneurocalcin varied across specific regions, but with the possible exception of the lateralstriatum, no obvious sex differences were apparent. Taken together, these data areconsistent with the idea that neurocalcin may be important for regulating sexualdimorphisms within the neural song system at a specific developmental period. Inaddition, because of variations in events related to post-transcriptional modification, athorough study of a gene's role in development and/or behavior should includeinvestigations of its transcription as well as translation.

© 2009 Published by Elsevier B.V.

Keywords:Song control systemImmunohistochemistrySex differenceApoptosismRNA–protein relationshipCalcium binding protein

1. Introduction

Songbirds such as the Australian zebra finch (Taeniopygiaguttata) possess a sexually dimorphic neural song circuit thatis associated with singing. This behavior is controlled by aseries of interconnected regions within the telencephalon.Area X and the lateral magnocellular nucleus of the anteriornidopallium (LMAN) are necessary for song learning (Bottjer etal., 1984; Scharff and Nottebohm, 1991). HVC (proper name)and the robust nucleus of the arcopallium (RA) form themotor

ney).

hed by Elsevier B.V.

pathway important for song production (Nottebohm et al.,1976; Simpson and Vicario, 1990). Several studies havedemonstrated that sexual dimorphisms exist at a number oflevels within this system (reviewed in Arnold, 1992; Wade,1999, 2001). For example, the volumes of HVC and RA aswell assoma size and numbers of neurons within these nuclei aregreater in males than in females. The projection from HVC toRA ismore robust inmales than in females. And Area X, whichis easily identified in males, is not visible in females usingstandard Nissl stains (Nottebohm and Arnold, 1976; Nixdorf-

Fig. 1 – Representative photomicrograph of neurocalcin-IRlabeling in HVC of amale (A) and female (B) at P18–23. Arrowsrepresent a sample of immunopositive neurons that wereidentified by a transparent nucleolus and cytoplasmicstaining that extended into processes. Scale bar=100 μm.

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Bergweiler, 1996). These neural dimorphisms begin to emergeby approximately post-hatching day (P) 8 and are completearound P60 (Bottjer et al., 1985; Kirn and DeVoogd, 1989;Nixdorf-Bergweiler, 1996). The exact mechanism(s) responsi-ble for creating these sex differences are not known; however,they are believed to involve a combination of hormonal and/orgenetic factors (Arnold, 2004; Arnold et al., 2004; Wade andArnold, 2004).

One particular gene product that may influence dimorph-isms within the song circuit is neurocalcin (Veney et al., 2003).Northern blot analyses of telencephalic tissue, using a specificprobe, detected three RNA species. Two of these productswereexpressed almost exclusively in females, primarily betweenP18 and P25 and not in adults of either sex. In agreement, insitu hybridization using a probe that recognized all threetranscripts indicated a broad butmore enhanced expression ofneurocalcin mRNA in females at P22 as compared to males.This is significant because we initially reasoned that thisincreased neurocalcin expression in females contributed tosexually dimorphic neural development by upsetting intra-cellular calcium [Ca2+]i homeostasis. Although the exactdetails are not known, according to the Ca2+ set pointhypothesis, proper cell functioning depends on a criticalwindow of [Ca2+]i (Johnson et al., 1992, Hwang et al., 1999).Conditions that result in prolonged elevated or low [Ca2+]ioutside of an established range can lead to cell death (Franklinand Johnson, 1992; Mattson, 1992; Yu et al., 2001; Case et al.,2007; Ringler et al., 2008). Consistent with this idea, many ofthe morphological differences in the song circuit of males andfemales are the result of enhanced cell death in females (Kirnand DeVoogd, 1989).

Unfortunately, very little is known about neurocalcin orexactly how it functions. This Ca2+ binding protein is amember of the visinin-like (VSNL) sub-family within theneuronal Ca2+ sensor proteins (Vijay-Kumar and Kumar,2002; Burgoyne, 2007; Braunewell and Klein-Szanto, 2009).VSNLs share a common four EF-hand motif; however, onlythree are functional (Braunewell and Gundelfinger, 1999;Burgoyne et al., 2004). As a whole, family members arebelieved to be involved in binding, buffering [Ca2+]i and/ormodulating the communication between Ca2+ and othercellular systems (Braunewell and Gundelfinger, 1999; Bur-goyne and Weiss, 2001; Burgoyne et al., 2004; Burgoyne, 2007).Neurocalcin has been identified in several species, cell types(neurons, interneurons and glia) and in various tissues. Forexample, in bovine it has been detected in several regions ofthe brain, adrenal glands and in cells of the retina (Nakanoet al., 1992; Terasawa et al., 1992; Hidaka and Okazaki, 1993;Nakano et al., 1993; Kato et al., 1998; Braunewell andGundelfinger, 1999). In rats, it is in the brain and cells ofthe auditory, visual and olfactory systems (Bastianelli et al.,1993, 1995a; Iino et al., 1995; Braunewell and Gundelfinger,1999). In humans, neurocalcin is present in the brain, ovariesand testes (Wang et al., 2001).

Generally, in order for a product to be important forsexually dimorphic development, it is assumed that expres-sion would be greater in one sex compared to the other duringthe critical period(s). Although neurocalcin mRNA was en-hanced in females, it was not clear how much of thatexpression would be represented by protein. Expression levels

of mRNA and protein exhibit a wide range of correlations fordifferent genes (Pascal et al., 2008). Studies indicate that thesecorrelation can range anywhere from 20% to 80% (Tian et al.,2004; Groves et al., 2006; Nie et al., 2006a,b). These data incombination with a recent study that demonstrated a sexdifference in neurocalcin protein expression in the mouseolfactory bulb (Murias et al., 2007) lead us to hypothesize thatneurocalcin protein is sexually dimorphic in the zebra finchbrain.

2. Results

Immunohistochemical labeling with a specific antibodyrevealed that neurocalcin protein was broadly distributedthroughout the brain. Cells of multiple shapes (fusiform,round and pyramidal) with an average soma size of 10–25μm were identified as immunopositive by cytoplasmic peri-nuclear staining that extended into processes (Fig. 1). For thequantitative analyses, neurocalcin-IR cells were counted in asingle representative section from each region. In RA therewas a main effect of age (F=3.97, p=0.031) and a significantinteraction between sex and age (F=6.04, p=0.007). Femaleshad more neurocalcin-IR neurons than males at P18–23. Inmales, neurocalcin-positive cells decreased from P10 to P18–23but increased again by adulthood (Fig. 2). In HVC, data revealeda marginally significant effect of sex (F=4.01, p=0.052), asignificant effect of age (F=4.76, p=0.018), but no interaction(F=2.20, p=0.132). Females had more neurocalcin-IR neuronsthanmales at P18–23. There was also a progressive increase inthe number of immunopositive cells detected in females, suchthat there were significantly more in adulthood than at P10(Fig. 3). For LMAN, there was a significant effect of age(F=71.48, p<0.001) but no main effect of sex (p=0.37) or an

Fig. 2 – Mean (±SEM) of neurocalcin-IR neurons in RA. Anasterisk (*) represents a significant sex difference at P18–23.In males, different letters denote a significant effect of agebetween groups. In females there was no significant effect ofage.

Fig. 4 – Mean (±SEM) of neurocalcin-IR neurons in LMAN.Within each sex, different letters denote a significant effect ofage.

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interaction (p=0.33). In both sexes, the number of cells thatcontained neurocalcin increased from P10 to P18–23 butdecreased by adulthood (Fig. 4). In Area X, statistical analysisalso revealed a significant effect of age (F=6.29, p=0.023). Thenumber of neurocalcin-positive neurons significantly de-creased from P10 to P18–23 and remained relatively un-changed through adulthood (Fig. 5).

We also investigated neurocalcin protein expression in asingle representative section of Rt, a monomorphic visualnucleus. As expected, there was no main effect of sex in thisregion, but there was a significant effect of age (F=23.06,

Fig. 3 – Mean (±SEM) of neurocalcin-IR neurons in HVC. Anasterisk (*) represents a significant sex difference at P18–23.In females, different letters denote a significant effect of agebetween groups. In males there was no significant effect ofage.

p<0.001). In males and females, the number of cells thatcontained neurocalcin remained relatively consistent at thetwo juvenile ages that were investigated but significantlydecreased by adulthood (Fig. 6). Interestingly, we also noticedthat out of all the regions that were quantitatively analyzed, Rtcontained the fewest number of neurocalcin-IR cells. For theneural regions that were semi-quantitatively analyzed, dataindicated that the relative number of immunopositive cellsvaried across specific regions. Based on visual observations,no obvious sex differences were apparent, with the possibleexception of the lateral striatumwhere the amount of labelingin males and females appeared different at each of the threeages that were examined (Table 1).

Fig. 5 – Mean (±SEM) of neurocalcin-IR neurons in Area X ofmales. Different letters denote a significant effect of age.

Fig. 6 – Mean (±SEM) of neurocalcin-IR neurons in Rt. Withineach sex, different letters denote a significant effect of age.

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3. Discussion

The present study documents a sex difference in the numberof neurocalcin-IR cells in two song production nuclei, RA andHVC, at P18–23. Based on counts taken from a singlerepresentative section, the female brain contained moreimmunopositive neurons than males. These data suggestthat within these areas, the dimorphic expression of neuro-calcin protein may influence physiological processes thatsignificantly contribute to the development of neural sexdifferences. The extent of the sex dimorphism was limitedonly to the neural regions that control song production. Astatistically significant sex difference was not detected inLMAN or the monomorphic visual nucleus Rt. This isinteresting because at this age range, males are forming

Table 1 – Relative amount of neurocalcin immunoreactivityin the zebra finch brain.

BrainRegion

P 10Male

P10Female

P18–22Male

P18–22Female

AdultMale

AdultFemale

A ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎AD ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎Cb ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎Hp ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎⁎ ⁎⁎⁎LSt ⁎ – ⁎⁎ ⁎ ⁎⁎ ⁎M ⁎⁎⁎ ⁎⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎N ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎NC ⁎⁎⁎ ⁎⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎⁎ ⁎⁎⁎POA/Hyp ⁎ ⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎ ⁎⁎TeO ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎

–=no detectable labeling; ⁎=low labeling; ⁎⁎=moderate labeling;⁎⁎⁎=high labeling. Abbreviations. A=arcopallium; AD=dorsalarcopallium; Cb=cerebellum; Hp=hippocampus; LSt = lateralstriatum; M =mesopallium; N = nidopallium; NC = caudalnidopallium; POA/Hyp=preoptic/hypothalamic area; TeO=optictectum.

mental templates of zebra finch song and have not yet enteredthe song vocalization phase (Immelmann, 1969). Based onvisual observations, there were also no apparent sex differ-ences in neurocalcin outside of song nuclei with the possibleexception of the lateral striatum. Although not statisticallyconfirmed, the significance of a sex difference in this region isnot immediately clear since to our knowledge it does not havesexually dimorphic characteristics associated with it. Further-more, the lack of an obvious protein sex difference in othermonomorphic regions of the brain (Table 1) served as a goodcontrol further strengthening the link between sex differencesin neurocalcin expression and developmental processes thatlead to dimorphic neural structures.

Across the brain regions that were quantitatively analyzed,data also indicated significant effects of age. Between P10 andadulthood, the number of neurocalcin-IR cells fluctuated inboth sexes. This finding is consistent with the idea that duringaging, region-specific dynamic changes in [Ca2+]i regulation isa normal part of the developmental process. For example, avariety of processes such as growth cone behaviors occur as aresult of varying [Ca2+]i levels (Mattson, 1992). In cultures ofembryonic rat hippocampal cells, neurite elongation andgrowth cone motility are suppressed at very low levels of[Ca2+]i (<50 nM). Slightly higher levels of [Ca2+]i (50–70 nM) willpromote the formation of microtubules but not microfila-ments. The developing axon can elongate but there will belittle growth cone motility. Further increases in [Ca2+]i (70–120 nM) permit neurite growth and growth cone motility.Functionally, Ca2+ binding proteins are necessary for regulat-ing [Ca2+]i for processes such as these. Therefore, maintainingthese critical windows of [Ca2+]i is crucial for normal celldifferentiation, function and survival. As long as the Ca2+

regulation occurs equally in both sexes, morphologicaldifferences are not likely to arise. However, if a range of [Ca2+]iis established but not regulated equally in both sexes,dimorphisms in various processes can result. More specifical-ly, when an established set point is exceeded or held belownormal levels for an extended period of time, Ca2+ homeosta-sis is disturbed and cellular structures and functions arecompromised, leading directly to cell death or an increasedsusceptibility to it (Johnson et al., 1992; Mattson, 1992;Braunewell and Gundelfinger, 1999; Hwang et al., 1999; Caseet al., 2007; Hara and Snyder, 2007). For example, over-expression of calcineurin promotes neuronal death in apo-ptosis-resistant cells (Jayaraman and Marks, 1997, 2000).Enhanced levels of calsenilin increase apoptosis in stable H4neuroglioma cells (Lilliehook et al., 2002). In the rat brain,dimorphisms in the expression of calbindin-D28k and calreti-nin during an early critical period affect cell death/survivalandmay be involved in sexual differentiation of hypothalamicstructures (Stuart and Lephardt, 1999; Brager et al., 2000).Taken together, these data support the idea that neurocalcinmay play a role in establishing sexual dimorphisms in theneural song system by altering Ca2+ homeostasis beyond anestablished set point during a specific period of development.

In addition to the above, several lines of evidence also pointto disturbances in Ca2+ binding proteins which may specifi-cally contribute to cell death associated with neurodegener-ative disorders. For example, in Alzheimer's disease (AD),neurocalcin, VILIP-1 and VILIP-3 are reduced in the temporal

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and entorhinal cortices (Shimohama et al., 1996; Braunewell etal., 2001). In contrast, another study found that calsenilin wasincreased in the cortex of Alzheimer's patients and in theneocortex and hippocampus of β-amyloid precursor proteintransgenic mice (Dong-Gyu et al., 2004).

The fact that neurocalcin proteinwas significantly increasedin females compared to males only at P18–23 is also consistentwith the idea that enhancedexpressionmaypromote cell death.Previouswork hasdemonstrated thatwhencompared tomales,the volumeof femaleHVCbegins to decrease as early as P10 andis statistically different by P20. In RA, the developmentaldivergence begins at P20 and is significantly different frommales by P30 (Nixdorf-Bergweiler, 1996).Differential cell death isthe primary mechanism responsible for creating neural sexdifferences within these regions (Kirn and DeVoogd, 1989). Notonly are the numbers of dying significantly increased at ageswhen a dimorphism in the volume of these nuclei are detected,but there is also an overlap with when we find neurocalcinprotein to be significantly increased in females. Interestingly,the sex difference in neurocalcin does not extend to earlier ages(P10) even though dimorphic neural events are taking place.Based on this, we conclude that neurocalcin is most likely notthe trigger that initiates dimorphic neural development butrather may play an important role during a limited specifiedperiod.

We did not detect a significant sex difference in neuro-calcin protein expression in LMAN, which parallels themorphological changes that are known to occur in thisnucleus during early development. In bothmales and females,the volume and the number of neurons in LMAN increase untilapproximately P20 and P35, respectively (Nixdorf-Bergweiler,1996; Nixdorf-Bergweiler and Von Bohlen Und Halbach, 2005).At P20, the volume of LMAN begins to first show a decrease inmales. Surprisingly, we did not detect a sex difference inneurocalcin protein in this region during this period (P18–23).This result can be explained if we consider the followingscenario. The maximum age of subjects within this groupingof animals was P23. Beyond this, we only investigated adults.Thus, there was about a 3-day window of overlap betweenwhen dying cells in LMAN are first detected in males andwhen we investigated neurocalcin protein. Depending on thedevelopmental age of the bird (as compared to the chrono-logical age), it is possible that we may have just missed thechange in neurocalcin expression. Perhaps if we had investi-gated animals at ages just beyond P23, for example at P25–P35,a sex difference in neurocalcin expression in this regionwouldhave become evident.

In general, we find that much of the mRNA expressiondescribed in Veney et al., (2003) was represented by functionalprotein. With the additional analysis of protein, a sexdifference in two song control regions emerged. Within cells,there is a dynamic relationship between mRNA and protein.At one time, a linear stoichiometric relationship was believedto exist. However, more recent studies indicate that thiscorrelation can range anywhere between 20% and 80%,indicating that mRNA and protein expression does not alwayscorrespond (Gygi et al., 1999; Chen et al., 2002; Tian et al., 2004;Nie et al., 2006a,b). There are several likely explanations thatcould account for this range of correlations. For example,translational activities can be partially or totally inhibited by

microRNAs (Farh et al., 2005; Lim et al., 2005; Baek et al., 2008;Bartel, 2009). Alternatively, the half-life of proteins and mRNAcan vary as a result of differences in the rate of degradation(Pratt et al., 2002; Carpousis, 2009). Based on these explana-tions, a comprehensive study of a gene should includeinvestigations on transcription, translation, degradation,posttranslational modification and function (Kasinath et al.,2008; Kozak, 2007).

To our knowledge, only one other study has reported a sexdifference in neurocalcin protein labeling (Murias et al., 2007).This paper examined expression across divisions of theolfactory bulb in mice and found a split between male- andfemale-biased dimorphic labeling which may contribute todifferences in olfactory processing related to sexual behavior.In addition to olfaction, neurocalcin has also been reported(although not dimorphically) in specialized cells within thevisual and auditory sensory systems (Nakano et al., 1992;Bastianelli et al., 1995b; Iino et al., 1995; Iino et al., 1998;Braunewell and Gundelfinger, 1999). In these systems and inthe brain, the exact mechanism of how neurocalcin regulates[Ca2+]i remains unclear, but there is likely some level ofcommonality. One possibility is that across systems, neuro-calcin affects ion channels, receptor function or membranetrafficking (Burgoyne et al., 2004). Under normal circum-stances Ca2+ binds neurocalcin and the complex activatescell specific membrane-bound cyclases, which in turn, reg-ulates activity of various cellular processes. Through feedbackproper levels of [Ca2+]i are maintained (Kumar et al., 1999;Krishnan et al., 2004; Braunewell and Klein-Szanto, 2009). Wepropose that when neurocalcin is dimorphically expressed,such as between P18 and P23 in our system, [Ca2+]i regulationis not within an established set point and cell death ensues.Current studies are underway to more completely character-ize this protein and its mechanisms of action.

4. Experimental procedures

4.1. Animals

Subjects were obtained from our facility at Kent StateUniversity. The animals were housed in communal aviariescontaining 5–7 pairs of breeders on a 14:10 L:D cycle. Adultswere fed a finch bird seed diet that was supplemented weeklywith hard boiled chicken eggs mixed with bread and freshoranges or spinach. Water and seed were provided ad libitum.Adequate measures were taken to minimize pain anddiscomfort to subjects. All procedures conformed to nationalguidelines and were approved by the Kent State UniversityAnimal Care and Use Committee.

4.2. Histology

Males (n=8) and females (n=8) were collected. We targetedanimals at ages P18–23. However, to investigate if neurocalcinmight affect brain development at other times, we alsoincluded animals from a time point earlier in development(P10) and adults (greater than P100). In all cases, subjects wereinjected with an overdose (0.10 mL) of equithesin anesthesiaand transcardially perfused with 0.75% phosphate-buffered

Fig. 7 – Control Western Blot verifying the specificity of theneurocalcin antibody. A single product corresponding to themolecular weight of neurocalcin (22 kDa) was detected inwhole telencephalic tissue.

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saline (PBS) followed by 150–200 mL of phosphate-bufferedformalin (PBF). Visual inspection of the gonads was used todetermine the animal's sex. The brain was extracted from theskull, post-fixed in PBF at room temperature for 2 h andcryoprotected in 20% sucrose at 4°C overnight. Brains werethen quickly frozen in dry ice and coronally sectioned at 30μmonto gelatin-coated slides. Consecutive slices were mountedonto alternate slides. The tissue was stored at −20°C untilprocessing.

For immunocytochemical labeling, sections were firstrinsed in 0.1 M PBS. To remove endogenous peroxidases, thetissue was then placed in 0.5% H2O2 in 0.1 M PBS for 15 minfollowed by 3, 5 min rinses in 0.1 M PBS. To minimize non-specific binding, 100 μL of 10% donkey serumwas added to thesections for 1 h. Neurocalcin delta primary antibody (Biomol;PlymouthMeeting, PA)was diluted 1:10,000 in 0.1MPBS, addedto the slides, and incubated at 4°C on a shaker for at least 48 h.After exposure to the primary antibody, slides were rinsed for5 min in PBS-T (0.4% Triton X-100). Biotinylated goat anti-rabbit secondary antibody (Vector Labs; Burlingame, CA)diluted 1:2000 was then incubated over the slides at roomtemperature for 1 h. Following the secondary, tissue wasbrieflywashed 3 times in PBS-T. Avidin–biotin complex (VectorLabs) was added to the slides at room temperature for 1 h andthen washed for 3 min in PBS-T. Neurocalcin protein wasvisualized after reaction with brown DAB (Sigma-Aldrich).

Specificity of the antibody was confirmed in two ways.First, we omitted the primary antibody. As a result, thecharacteristic labeling of the cytoplasm and cellular processeswere eliminated. Secondly, Western blot analysis of wholetelencephalon was performed. Briefly, tissue was removedand homogenized in 10 mL of RIPA (lysis) buffer with proteaseinhibitor for 1 h. Five micrograms of the protein sample wasloaded onto a gradient 4–15% SDS–PAGE ready gel (Bio-Rad;Hercules, CA) and transferred to a nitrocellulose membrane.After transfer, non-specific binding was blocked with 5% milk(2.5 g dry milk in 50 mL Tris-buffered saline). Neurocalcinprimary antibody diluted (1:5000) in 5% milk was incubatedover the membrane overnight at 4 °C. After a series of washes,horseradish-peroxidase-linked secondary antibody (goat anti-rabbit; 1:2000) was added to themembrane for 1 h. The proteinproduct was identified with a luminol peroxide solution (1:1)placed on themembrane. Chemoluminescencewas visualizedusing the Fujifilm LAS-3000. A single product that corre-sponded to the expected molecular weight of neurocalcinprotein was detected (22 kDa; Fig. 7).

4.3. Quantitative analysis of neurocalcin proteinexpression in song control nuclei

Based on previous work (Veney et al., 2003), there wasprecedence to believe that neurocalcin protein would heavilyoverlap with song control nuclei, but we did not know if theborders would be clearly defined. To aid in the identification ofregions,weutilized thionin-stained tissue.We selected a singlesection that corresponded to the approximate middle of HVC,RA, LMAN and Area X (where each nucleus would berepresented in its fullest cross-sectional extent). We alsoinvestigated labeling in a controlmonomorphic region, nucleusrotundus (Rt). Using MicroSuites Image Analysis (Olympus), a

digital image of the selected thionin-stained section wascaptured at 40× and overlaid with a digital image of neurocal-cin-IR labeling from an adjacent section at the same magnifi-cation. This technique allowedus to clearly identify the bordersof all nuclei. An observer whowas blind to the conditions of theexperiment counted the total number of neurocalcin immuno-reactive (−IR) neurons in the single representative tissuesection based on our established criteria (cytoplasmic peri-nuclear staining that extended into processes).

4.4. Semi-quantitative analysis of neurocalcin proteinexpression outside of song nuclei

In addition to song nuclei, neurocalcin mRNA was broadlydistributed in many other regions spanning the entire rostral–caudal continuum of the brain (Veney et al., 2003). We wereinterested in knowing how closely this reported expressionwould be represented by protein as well. To get an impressionof the overall distribution and relative amount of neurocalcinlabeling, specific neural regions were ranked. The assignedscores were based on a visual inspection of a single sectionfrom each region as described above. A “–” represented an areawith no detectable immunolabeled product, and at theopposite end of the spectrum, “⁎⁎⁎” was assigned to areaswith the highest amount of labeled cells. As in Veney et al.(2003), the following areas were analyzed; however, the nameshave been revised in the current report to reflect the updatedavian brain nomenclature (Reiner et al., 2004): arcopallium,

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dorsal arcopallium, cerebellum, hippocampus, mesopallium,nidopallium, caudal nidopallium, lateral striatum, preoptic/hypothalamic area and optic tectum.

4.5. Statistics

Two-way analyses of variance (sex×age) were employed todetermine statistical significance for measurements withinsong control regions and Rt. Data from Area X were analyzedby one-way analysis of variance. These were followed byTukey–Kramer post hoc tests for pairwise comparisons whensignificant main effects were observed. The level of signifi-cance was determined at p<0.05. All statistical analyses wereconducted using Sigma Stat. For the semi-quantitative rank-ings, data are presented descriptively.

Acknowledgments

The authors would like to thank Khadijah Wilson, Ann Dobry,Joshua Meeker and Kimberly Eustache for assistance with thisstudy. This work was supported by Kent State Universitylaboratory start-up funds and NINDS 1R15 NS067477-01 to S.V.

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