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Brain Research Bulletin 90 (2013) 132– 136

Contents lists available at SciVerse ScienceDirect

Brain Research Bulletin

jo u rn al hom epa ge : www.el sev ier .com/ locate /bra inresbul l

esearch report

orepinephrine inhibition in juvenile male zebra finches modulates adultong quality

uli Wadea,b,∗, Jennifer Lampenb, Linda Qib, Yu Ping Tanga,b

Michigan State University, Department of Psychology, East Lansing, MI 48824, United StatesMichigan State University, Neuroscience Program, East Lansing, MI 48824, United States

r t i c l e i n f o

rticle history:eceived 31 July 2012eceived in revised form 29 October 2012ccepted 31 October 2012

a b s t r a c t

During development, male zebra finches learn a song that they eventually use in courtship and defense ofnest sites. Norepinephrine (NE) is important for learning and memory in vertebrates, and this neuromodu-lator and its receptors are present throughout the brain regions that control song learning and production.The present study used the neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride

vailable online 15 November 2012

eywords:ongbirdevelopmentSP4

(DSP4) to reduce brain levels of NE in juvenile males. This manipulation inhibited the development ofquality songs, with some birds producing syllables that were unusually long and/or contained frequen-cies that were predominantly higher than normal. These results suggest that NE is important for theacquisition of typical song.

© 2012 Elsevier Inc. All rights reserved.

atecholamine

. Introduction

Norepinephrine (NE) plays important roles in vertebrate learn-ng and memory (Harley, 2004; Sara, 2009). Courtship song inasserine birds is a learned behavior. Juveniles acquire these voca-

izations from adult tutors, in many cases their fathers (Slater andann, 1990). The timing of this process and the degree of plastic-

ty that exists as animals mature varies among species. However,cross songbirds two key phases exist – a period of template for-ation in which a memory of tutor song is stored and a phase

f sensorimotor integration in which young animals match theireveloping vocalizations to the template (Marler, 1997; Nordeennd Nordeen, 1997).

Zebra finches are classic examples of ‘closed-ended’ or ‘age-imited’ learners. Memory acquisition begins at approximately 20ays post-hatching and continues to about day 60. Vocal practiceverlaps with this period, beginning at about 35 days of age andompleting around 3 months after hatching with the formation ofrystallized song. After this point, the song remains stable through-ut adulthood, in contrast to ‘open-ended’ learners in which songs

ay change in adulthood (Nordeen and Nordeen, 1997).Song is controlled by two interconnected circuits in the fore-

rain. An anterior pathway including Area X and the lateral

∗ Corresponding author at: Michigan State University, Neuroscience Program, 108iltner Hall, 293 Farm Lane, East Lansing, MI 48824-1101, United States.el.: +1 517 432 8301; fax: +1 5174322744.

E-mail address: wadej@msu.edu (J. Wade).

361-9230/$ – see front matter © 2012 Elsevier Inc. All rights reserved.ttp://dx.doi.org/10.1016/j.brainresbull.2012.10.010

magnocellular nucleus of the anterior nidopallium (LMAN) iscritical for song learning. A motor pathway important for the pro-duction of song consists of HVC (proper name; Reiner et al., 2004)projecting to the robust nucleus of the arcopallium (RA), whichinnervates the motoneurons of the vocal organ (syrinx; reviewedin Nordeen and Nordeen, 1997). The HVC and RA are substantiallylarger in males, who sing, compared to females who do not; Area Xis not visible in females (Wade, 2001; Wade and Arnold, 2004).

The locus coeruleus (LoC) is a prominent source of NE in thevertebrate brain. It has widespread projections in the forebrain ofbirds, including the song control nuclei. These brain regions con-tain high levels of NE, as well as �2- and �-adrenergic receptors.This pattern in adults is consistent with a role of NE in singing, andexperimental studies in adults suggest roles for NE in song produc-tion and perception (all reviewed in Castelino and Schmidt, 2010).In developing zebra finches, NE function in the song nuclei declinesafter 25–30 days post-hatching (Harding et al., 1998; Sakaguchi andSaito, 1989). This pattern is suggestive of a role for NE in templateformation. However, it is possible that activity of this neuromod-ulator peaks earlier, as measurements were not taken prior to day25.

To more directly test the role of NE in song systemdevelopment, we treated birds with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP4). This drug selectivelytargets axon terminals originating from the LoC and causes rapid

and substantial decreases in NE from this source (Jonsson et al.,1981). Our goal was to determine effects on development of bothsinging behavior (see above) and morphology of the forebrain songcontrol nuclei. These brain regions first become visible around 5–10

rch Bulletin 90 (2013) 132– 136 133

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Fig. 1. Representative spectrograms from each treatment group. Images depict twoseconds of song from animals administered DSP4 (top), zimelidine (middle), andsaline only (bottom). In the saline-treated bird, this clip contains 2 clearly identifi-able motifs. In the one administered zimelidine, roughly 2.5 motifs are included. Thepattern of notes in the DSP4 was less consistent, and the long notes (>300 ms) com-

J. Wade et al. / Brain Resea

ays after hatching (Gahr and Metzdorf, 1999; Kim et al., 2004;irn and DeVoogd, 1989). Maturation, which includes substantialrowth of these brain regions in males compared to females, occurshrough approximately day 50; sexual differentiation occurs at aigh rate between days 20 and 30 (Kirn and DeVoogd, 1989). Weodified the approach that was effectively used on adult male

ebra finches by Waterman and Harding (Waterman and Harding,008) and gave a first injection on post-hatching day 10 and aecond on day 18.

. Materials and methods

Animals. Experimental zebra finches were raised in adjacent walk-in aviariest Michigan State University, each containing roughly 5–7 breeding pairs and theirffspring, with 100 or more birds present in the room at any time. Animals werexposed to a 12:12 light:dark cycle, and provided ad libitum access to drinking water,eed (Kaytee Finch Feed; Chilton, WI), gravel and cuttlebone. Their diets were sup-lemented weekly with hard-boiled eggs, bread, spinach and oranges. Genetic sexf the birds was determined by PCR (Agate et al., 2002) on DNA extracted from toelips used as unique identifiers on the day of hatching. Only males were used in theresent experiment. All procedures were approved by the Michigan State University

ACUC and followed NIH guidelines.

.1. Treatment

On post-hatching days 10 and 18, birds were administered DSP4 (25 �g/g inaline, IP, Sigma–Aldrich, St. Louis, MO) or one of two control manipulations, ran-omly assigned across nests and aviaries. Because DSP4 can also be taken up byerotonergic cells, the selective reuptake blocker zimelidene dihydrochloride wasnjected one hour prior to prevent degradation of serotonerigic neurons ((Barclayt al., 1996; Waterman and Harding, 2008), 10 �g/g, IP, Sigma) in DSP4-treated ani-als. Controls consisted of injections on day 10 and 18 of either zimelidine followed

y saline one hour later, or two injections of saline an hour apart.

.2. Behavior

Birds were left to reach sexual maturity in their home aviaries, until 94–110 daysf age. To acclimate subjects prior to behavioral testing, each male was then housedn an individual cage in the same room as the colony aviaries for two days. They wereble to see and hear other birds within the room. The animals were then taken to aound-proof room and placed into an individual cage containing a sexually matureemale that they had not previously encountered. Sound was recorded for 1 h using aennheiser ME 62 onmidirectional microphone, Sound Devices USBPre2 amplifier,nd Adobe Audition software at a 44.1 kHz sampling rate, with 16-bit resolution.ecause audio but not video was recorded we cannot be sure, but these conditionsost likely resulted in the collection of female directed songs.

To obtain a rough estimate of relative levels of song learning, vocalizations ofxperimental males were compared to those of their fathers using Sound Analysisro 2011 software (Tchernichovski et al., 2000) by an observer blind to treat-ent group. Fathers were unambiguously determined by repeated observations of

ndividuals who shared the nest containing each hatchling. For father-son pairs, representative motif from each bird was compared. To assess song quality, apectrogram of a bout of song for each animal was analyzed based on the workf Simpson and Vicario (Simpson and Vicario, 1991). This method of selection wassed because a few birds sang a single bout, and because within birds that sang mul-iple bouts, they were very similar in content and structure (accuracy scores for eachairwise comparison for across motifs within a bout from one randomly selectedird per group ranged from 86.0 to 97.1, average = 91.4; accuracy scores across twoull bouts = 83.9–92.3, average = 88.7). Specifically, we determined whether unusualspects of the song existed based on definitions of Simpson and Vicario, includinghether the bout contained fewer than three distinct song elements (syllables),

nd syllables longer than 300 ms or predominantly higher than 1.5 kHz. In addition,he total number of syllables in the bout was counted. Finally, syllable duration andnter-syllable interval were determined for the bout using Adobe Audition software.verage values were calculated for each individual on these measures.

.3. Tissue collection and processing

If the male sang, it was rapidly decapitated within 30 min of the end of theecording, and its brain was removed and flash frozen in methyl butane. If the maleid not sing, it was returned in its individual cage to the colony room and recordedgain two days later. This process was repeated for a maximum of 5 trials. Anyirds that did not sing were euthanized at the end of the fifth recording session.

rains were stored at −80 ◦C until further tissue processing. The song of the fatherf each of the subjects was also recorded using the same protocol. Two of the fathersid not sing and were excluded from analysis along with their sons, leaving a totalf 22 subjects on which behavioral and brain data were analyzed: 7 treated withSP4 and zimelidine hydrochloride (hereafter identified as the ‘DSP4’ group), 7 with

bined with relatively limited breaks made it somewhat difficult to define discretemotifs. At least 4 notes in this clip (arrows) had elements that were predominantlyhigher than 1.5 kHz.

zimelidine hydrocholoride plus saline (‘zimelidine’), and 8 with two injections ofsaline (‘saline’).

Brains were coronally sectioned at 20 �m and thaw-mounted in 6 series ontoSuperFrost Plus slides (Fisher Scientific, Hampton, NH). Tissue was stored at −80 ◦Cwith dessicant until processing.

One series of slides from each animal was stained with thionin to facilitate iden-tification of brain regions and allow analysis of song system morphology. Anotherset was used for dopamine beta-hydroxylase (DBH) immunohistochemistry to iden-tify potential NE-containing cell bodies and fibers. DBH is the rate limiting enzymefor the conversion of dopamine to NE. All tissue was reacted simultaneously. Afterwarming to room temperature, it was rinsed in 0.1 M phosphate-buffered saline(PBS), fixed in 4% paraformaldehyde for 15 min, and washed 3 times in PBS (5 mineach). Slides were treated with 0.9% H2O2/methanol for 30 min and incubated for1 h in 10% normal goat serum in PBS with 0.3% Triton X-100. The tissue was thenincubated in a DBH rabbit polyclonal antibody (0.2 �l/ml; Cat# 22806, ImmunoS-tar, Hudson, WI) in 0.1 M PBS containing 0.3% Triton X-100, and 10% NGS overnightat 4 ◦C. This primary antibody was validated for use in songbirds (Castelino et al.,2007; Sockman and Salvante, 2008). A biotin-conjugated goat anti-rabbit secondaryantibody (0.5 �g/ml; Vector Labs, Burlingame, CA) was then used for 1.5 h at roomtemperature, followed by treatment with Elite ABC reagents (Vector Labs) anddiaminobenzidine (DAB) with 0.0024% hydrogen peroxide to produce a brown reac-tion product. Slides were then rinsed in PBS to be sure the reaction was terminated,dehydrated, and coverslipped with DPX (Sigma–Aldrich, St. Louis, MO).

2.4. Quantification of neural features

Analysis of brain tissue was completed by an observer blind to treatment group.Cross-sectional areas of HVC, RA and Area X were determined in each thionin-stained section in which it was visible using Image J software (National Institutes ofHealth). Brain region volume was calculated by summing the areas and multiplyingby the sampling interval. Measurements were taken on both sides of the brain, andan average was calculated, except in the few cases in which one side of the tissuewas damaged. In those instances, only the intact side was used.

DBH labeling was also quantified using Image J as a marker for relative NEexposure at the time behavior and morphology were evaluated. A 0.05 mm2 boxwas placed over the center of the LoC. Three or four locations were randomlyselected from the rostro-caudal extent of the nucleus and across the two sides of

134 J. Wade et al. / Brain Research Bulletin 90 (2013) 132– 136

Fig. 2. DBH immunohistochemistry in the song control nuclei (RA, Area X and HVC) and the locus coeruleus (LoC) of a saline-treated bird. The inset diagrams the location oft ng of

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did not sing; F2,13 = 1.88, p = 0.191 if these animals are excludedfrom the analysis). Syllable duration (F2,13 = 2.40, p = 0.130) andinter-syllable interval (F2,13 = 0.44, p = 0.656) were also consistentacross our manipulations (Table 1). Finally, treatment did not affect

Table 1Song characteristics across treatment groups. Values represent means (standarderror) calculated in ms from averages per individual.

he LoC at the level of the photograph, indicated with the arrow. Substantial labelias visible in each of the song control regions, with qualitatively more apparent

ongitudinalis medialis, TeO = optic tectum.

he brain, depending on tissue quality and availability (in a few animals the caudal-ost portion of the region was not collected). Immunohistochemical labeling was

ighlighted using the ‘threshold’ function (Huang setting), and the area coveredy cell bodies and fibers was calculated. Values were averaged within individuals.ibers within the song nuclei were not as abundant, and were classified on a 4 pointcale: 0 = none visible; 1 = very few fibers; 2 = moderate number of distinct fibers; 3ubstantial labeling of fibers.

.5. Statistics

Analyses reflected our primary interest of determining whether limiting expo-ure of the developing song system to NE modulates maturation of singing behaviornd/or song system morphology. One-way ANOVAs within each brain area weresed to determine whether DSP4 treatment affected the volumes of HVC, RA and/orrea X. They were also used to evaluate the indices of song quality and comparisons

o the father that were represented by continuous variables. We used a Chi-squareest to assess effects on the proportion of individuals with unusual syllables acrosshe groups. Finally, a one-way ANOVA was used to evaluate DBH labeling in the LoC,nd a Kruskal–Wallis test was employed to determine whether the extent of DBHbers differed across groups in adulthood. All analyses were computed using SPSSersion 19.

. Results

Of the 22 sons that were analyzed, 16 sang during the period of 5rials. The 6 that did not were from each of the three groups: 2 fromhe DSP4 group, 1 from the zimelidine treatment, and 3 adminis-ered just saline. Thus, juvenile treatment did not appear to affect

cell bodies and fibers was observed in the LoC. Punctuate labeling typical of fibers HVC, and particularly RA, compared to Area X. Cb = cerebellum, FLM = Fasciculus

whether an individual sang under these testing conditions. How-ever, it did affect song quality. Of the 16 individuals that sang, onlybirds that were administered DSP4 produced unusual syllables (asdefined by (Simpson and Vicario, 1991)). Three of 5 sang long notesand one of these also sang notes with unusually high frequencies(Fig. 1). These results are in contrast to 0 of 6 in the zimelidineand 0 of 5 saline groups producing these types of unusual syllables(X2 = 8.12, p = 0.017).

All birds that sang, with the exception of one in the zimeli-dine group, produced more than 3 distinct song elements. Thetotal number of syllables in the bouts did not differ across thegroups (F2,19 = 0.62, p = 0.548 if zeros are included for birds that

Treatment Syllable duration Inter-syllable interval

DSP4 142.8 (8.7) 30.2 (2.6)Zimelidine 134.4 (10.5) 32.4 (4.9)Saline only 112.4 (9.7) 36.9 (6.5)

rch Bulletin 90 (2013) 132– 136 135

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J. Wade et al. / Brain Resea

ny of the variables evaluated across the father and son songsn Sound Analysis Pro: similarity (F2,13 = 0.34, p = 0.715), accuracyF2,13 = 0.32, p = 0.732), sequential matches (F2,13 = 0.28, p = 0.759),itch (F2,13 = 1.04, p = 0.380), wiener entropy (F2,13 = 1.90, p = 0.188),requency modulation (F2,13 = 0.81, p = 0.467), amplitude modu-ation (F2,13 = 2.18, p = 0.153), or goodness of pitch (F2,13 = 0.12,

= 0.883).The general pattern of DBH labeling across the brain paralleled

revious work (Mello et al., 1998). Qualitatively, Area X consis-ently contained fewer DBH+ fibers than HVC and especially RAFig. 2), but relative levels of expression within these song nucleiid not differ across the treatment groups (Kruskal–Wallis all

> 0.300). Similarly, LoC labeling was consistent across treatmentsF2,18 = 0.43, p = 0.656). These results are consistent with the ideahat the DSP4 manipulation during development did not have per-

anent effects on catecholamine levels either at their source or inhe forebrain song control regions.

Juvenile treatment with DSP4 had no effect on the adult volumef HVC, RA or Area X (all F2,16–19 < 1.40, p > 0.293; Fig. 3).

. Discussion

DSP4 treatment of juvenile male zebra finches inhibited theevelopment of typical song in some individuals; it induced a deficit

n the quality of songs produced in adulthood. This result suggestshat early exposure to NE is important for the establishment oformal vocalizations. The DSP4 manipulation did not affect the vol-me of the song control nuclei, which indicates either that NE isot critical for maturation of this feature or that the size of theseegions can still reach control values even if development was tem-orarily inhibited while NE levels were low. We believe the latter

s more likely, as pilot data indicated that DSP4 caused a reduc-ion in the size of song control regions. These birds were treatedn a manner similar to the present study, with injections of zimeli-ine hydrochloride and DSP4 on post-hatching days 8 and 18. Theyere euthanized on post-hatching day 25, one week following the

econd set of injections. Quantification of Nissl-stained sections inirds treated from DSP4 (n = 3) and unmanipulated males (n = 4)

ndicated a 40% decrease in the volume of HVC (t5 = 13.3, p < 0.001)nd a 15% decrease in the volume of RA (t5 = 2.98, p = 0.031) due toreatment. Area X was 25% smaller in DSP4- compared to saline-reated birds, but this effect did not reach statistical significance,erhaps because tissue was damaged in one control animal so theample size was only 3 (t4 = 2.22, p = 0.091). In contrast to controls,BH labeling in the LoC was limited in two and not detectable in

he other one of these DSP4-treated birds.We do not know exactly how long our manipulation was

ffective, although NE projections appeared to have recovered bydulthood, as indicated by equivalent DBH labeling in the songuclei across the groups. Prior to the present study, DSP4 in zebranches had only been used in adults. Intracerebroventricular injec-ions resulted in inhibition of NE compared to control treatment ineveral regions of the forebrain, including Area X and LMAN, 10ays later (Barclay et al., 1996). Systemic injections also inducedubstantial reductions in NE in regions including Area X and RA40–43%), but these were not significantly different from controlsBarclay et al., 1992). Using manipulations that more closely par-lleled those in the present study (two injections 10 days apart),BH-labeled neurons in the LoC were decreased by 60% quanti-ed 10 days following the second injection. This DSP4 treatmentaused significant reductions in the percent area with DBH label-

ng in HVC, RA and LMAN (Waterman and Harding, 2008). Studiesn rodents have produced variable results from peripheral injec-ions of DSP4, but generally document inhibition of a duration thatould cover much, if not all, of song system development. These

reduced in some groups due to tissue damage that prevented accurate tracing of theborders in some sections. No significant effects were detected.

include effects in the cortex of projections beginning to recoverat 3–4 weeks and becoming comparable to controls at 4–6 months(Fritschy and Grzanna, 1992), NE concentration significantly dimin-

ished at 8 months and taking a year for full recovery (Wolfman et al.,1994), and a reduction of about 70–80% within 6 h and as much as95% at two weeks, with effective reduction for at least a month(Grzanna et al., 1989; Jonsson et al., 1981).

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We also do not know where in the brain reduced NE may haveffected song development. In addition to song control nuclei, NEs present in auditory areas of the forebrain, including the caudo-

edial mesopallium (CMM) and caudomedial nidopallium (NCM)here song templates may be stored, as well as the preoptic areahich is involved in motivation to sing (all reviewed in Castelino

nd Schmidt, 2010). Work in brain slices from juveniles suggestshat NE modulates LMAN input to RA (Sizemore and Perkel, 2008),ircuitry critical for vocal learning.

It is difficult to draw conclusions about song learning specificallyrom this study. Songs might have been more alike if we had isolatedather/son pairs (Tchernichovski and Nottebohm, 1998). However,e felt it was important to test the effects of NE depletion in theore natural social environment of a colony (Zann, 1996). Under

hese types of conditions in the laboratory, males learn from mul-iple tutors (Williams, 1990). Thus, it is not necessarily surprisinghat similarity scores were highly variable (ranges: DSP4 = 22–88,imelidine = 14–68, saline 18–57). However, these and all otheromparative measures between father and son were equivalentcross the groups. These results suggest that inhibition of NE duringevelopment may not specifically diminish the potential for a maleo copy features of his father’s song, but more likely influences theddition of unusual elements.

While our temporary inhibition of NE did not affect develop-ent of typical masculine volumes of the song control nuclei, the

euromodulator does play a role in sexual differentiation of theodent brain. NE mediates the masculinizing effects of estradioln the preoptic area, including increasing the size of the sexuallyimorphic nucleus of the preoptic area (SDN-POA), which is larger

n males than females. It may also be involved with defeminizationr feminization of behavior, depending on the receptors at whicht acts [reviewed in Wilson and Davies, 2007].

In conclusion, the present study provides the first in vivo exper-mental evidence that NE is involved in song development in theebra finch. This work complements a body of descriptive evidencen the distribution of NE, DBH, and relevant receptors, as well asunctional evidence of roles of NE in adult song production and per-eption (Castelino and Schmidt, 2010). Future work should utilizepecific agonists and antagonists across key stages of morpholog-cal and functional development to assess the roles of NE in moreepth.

cknowledgments

We are grateful to the undergraduates in the lab who helpedith animal care, to Camilla Peabody for sexing the birds, andhelsea Hatcher and Sara Weiss for assistance with song recordings.his work was supported by NIH grant MH55488.

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