Reduced Vocal Variability In A Zebra finch Model Of .
Physiological Reports ISSN 2051-817XORIGINAL RESEARCHReduced vocal variability in a zebra finch model ofdopamine depletion: implications for Parkinson diseaseJulie E. Miller1,*, George W. Hafzalla2,*, Zachary D. Burkett2, Cynthia M. Fox3 & Stephanie A. White21 Departments of Neuroscience and Speech, Language and Hearing Sciences of the University of Arizona, Tucson, Arizona2 Integrative Biology & Physiology, University of California, Los Angeles, California3 National Center for Voice and Speech, Denver, ColoradoKeywords6-hydroxydopamine, Parkinson disease,songbird, zebra finch.CorrespondenceJulie E. Miller, Department of Neuroscience,University of Arizona, 1040 E 4th St. GS 611,Tucson, AZ 85716.Tel: (520) 626-0100Fax: (520) 621-8282E-mail: juliemiller@email.arizona.eduFunding InformationFunding provided by NIH R03NS078511(awarded to JEM and transferred to SAW),the Parkinson’s and Movement DisorderFoundation (JEM), and the AmericanParkinson Disease Association (SAW).Received: 1 October 2015; Accepted: 6October 2015doi: 10.14814/phy2.12599Physiol Rep, 3 (11), 2015, e12599, doi:10.14814/phy2.12599AbstractMidbrain dopamine (DA) modulates the activity of basal ganglia circuitryimportant for motor control in a variety of species. In songbirds, DA underlies motivational behavior including reproductive drive and is implicated as agatekeeper for neural activity governing vocal variability. In the zebra finch,Taeniopygia guttata, DA levels increase in Area X, a song-dedicated subregionof the basal ganglia, when a male bird sings his courtship song to a female(female-directed; FD). Levels remain stable when he sings a less stereotypedversion that is not directed toward a conspecific (undirected; UD). Here, weused a mild dose of the neurotoxin 6-hydroxydopamine (6-OHDA) to reducepresynaptic DA input to Area X and characterized the effects on FD and UDbehaviors. Immunoblots were used to quantify levels of tyrosine hydroxylase(TH) as a biomarker for DA afferent loss in vehicle- and 6-OHDA-injectedbirds. Following 6-OHDA administration, TH signals were lower in Area Xbut not in an adjacent subregion, ventral striatal-pallidum (VSP). A postsynaptic marker of DA signaling was unchanged in both regions. These observations suggest that effects were specific to presynaptic afferents of vocal basalganglia. Concurrently, vocal variability was reduced during UD but not FDsong. Similar decreases in vocal variability are observed in patients withParkinson disease (PD), but the link to DA loss is not well-understood. The6-OHDA songbird model offers a unique opportunity to further examine howDA loss in cortico-basal ganglia pathways affects vocal control.*Both authors contributed equally to thework.IntroductionSongbirds offer attractive models for investigation ofbrain–behavior relationships at gene, circuit, and organismal levels. They share similar reciprocally connected cortico-striatal loops with mammals, but offer the additionaladvantage of a well-characterized neural circuitry forvocalization. Critically, the loci for song production areneuroanatomically distinct and well-characterized including Area X, the specialized subregion of the songbirdbasal ganglia dedicated to vocal learning and maintenance(Fig. 1A). As in mammals, the basal ganglia receivedopaminergic innervation from the midbrain ventraltegmental area (VTA) and substantia nigra pars compacta(SNc) (Bottjer 1993; Lewis et al. 1981). Feedback loopsexist between these regions and Area X, as in mammalianstriatum (Person et al. 2008; Gale and Perkel 2010).Dopamine (DA) orchestrates a delicate balance withinmammalian and songbird basal ganglia circuits in processesassociated with motor exploration versus performance andreward-based behavior (Shiflett and Balleine 2011). Thesongbird model enables further exploration of the role ofDA in these processes, given that it contains medium spinyneurons (MSNs) and globus-pallidal neurons in the basalª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf ofthe American Physiological Society and The Physiological Society.This is an open access article under the terms of the Creative Commons Attribution License,which permits use, distribution and reproduction in any medium, provided the original work is properly cited.2015 Vol. 3 Iss. 11 e12599Page 1
J. E. Miller et al.6-OHDA Injection into Area X Reduces Song VariabilityAHVCLMANRAXDLMSNc/VTAnXIItsSyrinx & TracheaBKEYDay 0morningDay 0afternoonDay 4morningDay 50 hour NSFDPre UD/FD Area X Injections6-OHDA or VehicleUDPost UD/FD EndpointBrain RemovedNSCNXXVSPNVSP1mmFigure 1. Neuroanatomy of the song circuitry and experimentaltimeline. (A) The song control system mainly consists of twointerconnected loops: the vocal production pathway (solid lines)containing cortical nuclei HVC (proper name) and the robustnucleus of the arcopallium (RA); the anterior forebrain pathway(dashed lines) including basal ganglia Area X (X), the dorsolateraldivision of the medial thalamus (DLM) and the cortical lateralmagnocellular nucleus of the anterior nidopallium (LMAN). Area Xreceives DA input (dotted arrow) from the substantia nigra parscompacta (SNc) and ventral tegmental area (VTA) (Gale and Perkel2010). A dotted line indicates the coronal plane of section shownfor (C). Modified from (Miller et al. 2008). nXIIts –tracheosyringealportion of the hypoglossal motor nucleus. Other abbreviations intext. (B) Experimental timeline. The key (Fig. 1B), represents thebehavioral contexts for 2 h of undirected (UD) song, femaledirected (FD) song and 0 h non-singing (NS), the experimentalendpoint. In the case of insufficient singing, this timeline wasadjusted 1 day for pre-surgery and post-surgery song collection.(C) Schematic of male zebra finch coronal brain section (left)indicates anatomical regions and micropunches in the thioninstained section (right).ganglia, both of which are DA-sensitive (Simonyan et al.2012). Both cell types share similar anatomical and physiological signatures with their mammalian counterparts (Farries and Perkel 2002; Goldberg and Fee 2010). Levels of DAin the songbird basal ganglia are closely associated withsocial and breeding contexts. For example, in Area X ofmale European starlings and zebra finches, DA levelsincrease in the presence of a conspecific female, underlyinghis motivation to sing, as assessed through measurementsof the rate-limiting catecholamine biosynthetic enzyme,tyrosine hydroxylase (TH) (Heimovics and Riters 2008)2015 Vol. 3 Iss. 11 e12599Page 2and microdialysis of DA metabolites (Sasaki et al. 2006). Incontrast to the female-directed (FD) song, DA levels arelower during undirected (UD) song, when the male singsalone (Hara et al. 2007). DA cells in the VTA provide onesource of neuromodulation onto MSNs to regulate thesesocial-context-dependent singing behaviors (Yanagiharaand Hessler 2006). Targeting these Area X inputs usingneurotoxins can yield insight into the neuromodulation ofvocal behavior.In rodents, injecting the neurotoxin 6-hydroxydopamine (6-OHDA) into either the medial forebrain bundleor striatum poisons DA nerve terminals measured by THimmunostaining. This results in motor phenotypescharacteristic of Parkinson disease (PD) with alteredultrasonic vocalizations detected at 72 h and 4 weekspost-injection (Grant et al. 2015). In zebra finches, a unilateral injection of 6-OHDA into the VTA/SNc reducesTH immunostaining in Area X (Hara et al. 2007). Consequently, FD song becomes slower. No overt changes inmotif structure were noted, but acoustic variations inindividual syllables were not assessed.Here, 6-OHDA was injected directly into Area X toassess consequences on DA biomarkers and song. Wehypothesized that 6-OHDA administration to Area Xwould reduce DA signal during both UD and FD songand lead to changes in song features. We also predictedthat UD would be more sensitive to DA depletion giventhat levels are relatively low under this condition (Haraet al. 2007); further reduction could drain any reservoirof signal. In normal adult males, higher levels of DA during FD song (Sasaki et al. 2006) are associated with lessvocal variability but when D1 receptors are blocked, FDsong resembles the more variable UD song (Leblois et al.2010). Similarly, we predicted that with experimental DAdepletion following 6-OHDA injection, acoustic featuresof FD song would resemble UD. To measure changes inDA signal in the basal ganglia, TH and dopamine receptor-associated postsynaptic protein (DARPP-32) levelswere quantified using an approach, not feasible in rodentmodels, of separately micro-punching vocal versus nonvocal subregions followed by immunoblotting for DAbiomarkers. Below, evidence is provided for depletion ofpresynaptic DA terminals specifically within vocal Area Xand associated changes to UD, but not FD, song. Noapparent effect of experimental DA depletion in the socialcontext was detected.MethodsSubjectsAll animal use was approved by the Institutional AnimalCare and Use Committee at the Universities of Californiaª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf ofthe American Physiological Society and The Physiological Society.
J. E. Miller et al.Los Angeles and Arizona. For the experiments 25 birdswere used, including a subset for tissue analyses. Adultmale zebra finches (120–400 days) were moved to individual sound attenuation chambers and acclimated undera 13:11 h light:dark cycle. Behavioral experiments wereconducted in the morning from lights-on until overdosewith inhalation anesthetic. Brain tissue for immunoblotting and immunohistochemistry was collected from birdsthat were euthanized immediately following lights-on (0 hnonsinging, NS, Fig. 1B) to prevent any confound ofbehavioral context and/or circadian changes.BehaviorMethods followed those of Miller et al. (2008) with sometime-course modifications (Fig. 1B). UD song recordingwas ongoing. FD song was captured on the day prior toor the morning of surgery and on day 4 or 5 postsurgery, depending on when singing levels were sufficient.Non-vocal behavior was simultaneously video-taped preand post-treatment during song recording. An experimenter blind to the treatment scored the pre versus postsurgery occurrence of non-song behaviors during 30 minof UD and FD song beginning at lights-on.Song recording and analysisSounds were recorded (Shure SM58/93 microphones) anddigitized (PreSonus Firepod/Audiobox: 44.1 kHz samplingrate/24 bit depth). Recordings were managed using SoundAnalysis Pro (SAP) (Tchernichovski et al. 2000).The song was hand-segmented following Miller et al.(2010). Motifs were identified as a repeated order of multiple syllables, excluding introductory notes and unlearnedcalls. Syllables were identified as sound envelopes thatcould be separated from other syllables by local minima.WAV files from 20 consecutive renditions of motifs andsyllables during UD and FD songs were selected (Audacity, audacityteam.org) from a similar morning timepoint and run in SAP for measures of self-accuracy andindividual acoustic features (SAP manual). The meanwith standard error (SEM), and coefficient of variation(CV) of each feature are reported in Table 1 with poweranalysis reported for each acoustic feature. Syllable typesknown as harmonic stacks were analyzed separately forchanges in fundamental frequency (FF) variability (Kaoet al. 2005) using code provided by M. Brainard, UCSF(MATLAB, Mathworks, Natick, MA).Statistics and data presentationFor the means and CVs of all syllable acoustic featuresand self-similarity scores, resampling one-way ANOVA6-OHDA Injection into Area X Reduces Song Variabilityindicated a significant (P 0.05) within-bird syllableeffect. Thus, syllables were treated as independent of eachother (Figure S1 and text). No appreciable increase inpower was observed in any statistical test when conductedon an n 20 syllables in a given behavioral condition(Miller et al. 2010). Therefore, all analyses were conducted on the first 20 syllable renditions/session.Song features are presented in Figures 4–7C as effectsizes. Effect size was calculated using the formula(AB)/(A B), where A and B are measures of a givenfeature in two different conditions, respectively (e.g.,before vs. after surgery or in FD vs. UD song). For theeffects of surgery (Figs. 4–6A), negative values indicatethat a given metric is greater before injection of the drugor vehicle whereas positive values indicate the opposite.Values near zero indicate no surgical effect. For the effectof social context (Figs. 6B and 7C), negative bars indicatethe value is greater in UD. “Pre” denotes all pre-surgerysyllables regardless of the treatment that the bird received.“Vehicle” are comparisons between post-vehicle FD andpost-vehicle UD and “6-OHDA” are comparisons betweenpost-6-OHDA FD and post-6-OHDA UD.Effect size calculations enable statistical comparisonacross treatment groups or social contexts (i.e., 6-OHDAvs. vehicle-injected birds; UD versus FD), which is notpossible using the traditional pre versus post-paired plotcomparisons done within a treatment group. Because thiscalculation normalizes the scores for each measure, it isnot influenced by between-syllable differences. It alsoallows a single number to represent the effect of a condition on a syllable feature. An unpaired resampling test onthe median for each measure evaluated one condition’seffect size against another’s and is reported in the text.Since the effect size calculation places all data on a scaleof 1 to 1, we also report all raw untransformed meansand SEM in Table 1 with P-values generated from theraw scores. A significant effect of 6-OHDA injection wasassessed when a P-value 0.05 was attained for the 6OHDA group and not in the vehicle group.Figures S2–S3 show the more conventional paired plotsof the coefficient of variation (CV) of fundamental frequency, presented similarly to Kao et al. (2005). Forexample, song feature scores were collected for a group ofsyllables before (pre) and after (post) surgical delivery ofvehicle or 6-OHDA. All syllables from animals injectedwith vehicle were then viewed as pairs of pre-vehicle andpost-vehicle data, and the statistical significance of thechange resulting from vehicle injection was assessed. A Pvalue is determined by resampling the data to repeatedlysimulate the null hypothesis in order to see how likely adifference of the magnitude observed would occur bychance. A similar calculation was performed for the syllables of all animals injected with 6-OHDA. A significantª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf ofthe American Physiological Society and The Physiological Society.2015 Vol. 3 Iss. 11 e12599Page 3
2015 Vol. 3 Iss. 11 e12599Page 929.7711.7870.12031.87882.72310.5840.2440.1670.162 0.033 0.190 0.0550.519 0.2770.057 0.601––––––SEMCVSEMStats0.065 0.007 0.058 0.0090.082 0.007 0.085 0.0070.062 0.004 0.057 0.0050.089 0.006 0.089 0.0060.168 0.053 0.175 0.0380.032 0.003 0.030 0.002––0.065 0.006 0.054 0.0050.092 0.008 0.083 0.006––Power0.275 0.2650.550 0.1260.160 0.3050.875 0.1160.882 0.1590.330 0.242––0.052 0.5890.050 0.5420.074 0.004 0.060 0.003 0.001 0.9990.096 0.007 0.089 0.006––0.499 047Mean1.3890.09824.86938.6332.392341.5007.967 0.2780.184 0.2981.385 0.2000.097 0.25325.536 0.8779.5960.1700.188119.47094.59198.2489.538 0.8590.147 0.0600.151 0.8771.5300.10925.07636.7972.476314.1561.631 0.2130.110 0.00624.651 6––SEM––CVPost––SEM––0.160 0.027 0.127 0.022 057 0.005 0.054 0.004 0.4490.081 0.004 0.084 0.005 0.4490.061 0.004 0.063 0.005 0.5700.099 0.008 0.094 0.007 0.4190.126 0.032 0.113 0.024 0.7080.053 0.006 0.050 0.005 0.330––0.055 0.005 0.060 0.004 0.3450.080 0.004 0.081 0.005 0.6750.072 0.004 0.074 0.004 0.5480.093 0.005 0.097 0.006 ––0.2560.1390.1540.2010.3640.097––P-value PowerStats0.042 0.004 0.043 0.004 r3349.944 112.649 3300.613 110.570 0.16037.3442.405318.4141093.499 186.007 1142.788 185.375 0.448119.21194.85098.275PreP-value Power CV0.217 0.100SEM3398.027 101.422 3423.937 101.758 MStats1187.255 164.738 1131.548 163.828 0.376122.79094.033MeanPower––Power MeanMeanSEM0.364 0.2320.586 0.1490.043 0.5270.021 0.7760.834 0.1180.284 0.2690.274 0.6430.612 0.3600.060 0.5680.006 0.7900.103 0.405––P-valueVehicleSEM0.556 0.1220.057 0.455––0.032 0.003 0.030 0.003––CV6-OHDAMean0.015 0.7260.128 0.425Power CV9.914 0.001 0.8970.2070.156P-valuePostMean, standard error (SEM), P-values, and power analyses are reported for pre versus post-surgery comparisons for the 6-OHDA and vehicle-injected bird groups for undirected (UD) andfemale-directed (FD) song. Significance scores are highlighted by their P-values obtained from resampling paired-tests on individual syllables pre versus post-surgery. Highlighted (in bold) Pvalues are shown for mean accuracy, syllable duration, FM, and decreased Wiener entropy CV for UD song post-6-OHDA 67.609115.67894.07797.971Mean3563.837 135.539 3526.234 134.62136.975Flat harmonic syllables 7410.2590.204SEM3617.879 113.442 3666.710 e6-OHDATable 1. Summary of song features.6-OHDA Injection into Area X Reduces Song VariabilityJ. E. Miller et al.ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf ofthe American Physiological Society and The Physiological Society.
J. E. Miller et al.effect of 6-OHDA injection was determined by observinga statistically significant difference in the 6-OHDA datapairs that was not present in the vehicle data pairs.For DA biomarkers, a resampling unpaired differencetest was used to detect group differences and confirmedby a Mann–Whitney U test. Resampling was also used toassess the treatment effect (vehicle vs. 6-OHDA) on nonsong behavior. For a complete description of the use ofthe resampling method for birdsong analysis and relatedcitations, refer to Miller et al. (2010) and Burkett et al.(2015).Surgical procedure and drug dosagesSurgery was conducted on isoflurane-anesthetized birds(n 11, bilaterally-injected vehicle birds; n 9, 6-OHDAbirds; n 3, received unilateral injections of 6-OHDAand vehicle within the same bird). A glass pipette was fitted into a Nanoject II pressure-injector and back-filledwith mineral oil then loaded with either 0.1% sodium Lascorbate in ddH20 (Sigma #A7631, vehicle) or 6-OHDA(Sigma, lots #MKBPO832V, #MKBR6609V) dissolved invehicle. A 1.2 lg bilateral dose of 6-OHDA reliably sparedthe integrity of Area X while leading to subtle effects onsong (see Results). Pilot work on the optimal dose to elicit changes in song while preserving Area X integrity indicated that variability in potency across lots of 6-OHDAnecessitates testing of each new lot. In pilot work, ahigher dose of 4 lg resulted in the same changes in UDsong (Miller et al. 2009) reported in this current study atthe 1.2 lg dose. However, following this work, a range of2–4 lg doses of 6-OHDA induced a lesion in Area X thatwas not due to electrode damage because Area X of vehicle-injected birds remained intact. Studies of rodent models of 6-OHDA have also reported these deleteriouseffects, using a 6–8 lg dose (M. Ciucci, pers. comm.2010-2011). Given these considerations, each new vial ofdrug was tested. Lower doses of 6-OHDA (0.6, 0.8 lg)failed to yield detectable changes in song (data notshown) even though reduced TH levels were observed inimmunoblots with as little as 0.6 lg (Fig. 2B).To prevent oxidation, 6-OHDA was prepared within30 min of use and kept covered on ice to minimize lightexposure. Area X was targeted from the bifurcation ofthe mid-sagittal sinus, in mm: 5.15 rostral, 1.5–1.6 lateral,and a depth of 3.0–3.3. Injections were delivered every15 sec. The total volume was 250 nL for 6-OHDA(n 9) and 250 or 500 nL for vehicle (n 11). No effectof vehicle volume on song features was observed, so vehicle-injected birds were pooled. After 5 min, the pipettewas slowly retracted and the tip visually inspected forclogging. Following post-operative monitoring, birds werereturned to their chambers and recorded until death.6-OHDA Injection into Area X Reduces Song VariabilityTissue preparation and immunoblottingBilateral micropunches of Area X and outlying VSP andnidopallium (N; Fig. 1C) were obtained, processed andimmunoblotted according to Miller et al. (2008) but witha PVDF membrane. Post hoc thionin staining of punchedsections enabled verification of their anatomical precision(Fig. 1C). DA biomarkers (Figs. 2, 3) were detected withovernight incubation at 4 C with primary antibodiesagainst TH (Millipore #AB152, rabbit 1:500, 1:1500 andDARPP-32 Abcam #ab40801, rabbit 1:10,000, 1:30,000dilution; Murugan et al. 2013). A primary antibody toGAPDH (Millipore #MAB374, mouse 1:10,000) served asa loading control because neither the Area X protein normRNA levels are affected by this behavioral protocol(Miller et al. 2008; Hilliard et al. 2012a). Following TBSTwashes, blots were probed with HRP secondary antibodies: anti-rabbit IgG (1:2000 – TH, 1:10,000 – DARPP-32)and anti-mouse IgG (1:6000–1:10,000 – GAPDH; Amersham Pharmacia Biotech) for 2 h at room temperaturethen washed. Blots were developed using chemiluminescence and imaged (Typhoon scanner, or Bio-Rad system)with quantification done in Quantity One (Bio-Rad) byan experimenter blind to the behavioral condition. Densitometric analysis of bands on the immunoblots was aspreviously described (Miller et al. 2008; Hilliard et al.2012a). Briefly, a rectangular band was drawn to encapsulate the signal of interest deemed a “raw” value (the “volumetric” measurement in Quantity One) and a same-sizerectangular band was placed in the lane above or belowthe band to subtract the “background” signal. Thisyielded a corrected value. Corrected values were obtainedfor TH, DARPP-32 and GAPDH. Corrected values forTH and DARPP-32 were then divided by a correctedGAPDH value per lane to control for equal protein loading. Protein values reported in Figures 2–3 represent thesenormalized values. Results were independently confirmed,using NIH Image J and the same procedure above wasbased upon densitometric measurements of the bands.ImmunohistochemistryThree adult male zebra finches were injected with 1.2 lgof 6-OHDA in Area X of one hemisphere and with vehicle in the other. Brains were collected following a transcardial perfusion of warmed saline followed by 4% roomtemperature paraformaldehyde in Phosphate Buffer Saline(PBS) on the morning of day 5 (0HR NS). Fixed brainswere cryoprotected in 20% sucrose overnight, then frozenin dry ice and sectioned at 30 lm on a cryostat(Microm). The targeting of Area X was visually verifiedwhile sectioning by identification of the electrode track.The injection of 6-OHDA results in a brown discolorationª 2015 The Authors. 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6-OHDA Injection into Area X Reduces Song Variability2015 Vol. 3 Iss. 11 e12599Page 6J. E. Miller et al.ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf ofthe American Physiological Society and The Physiological Society.
J. E. Miller et al.6-OHDA Injection into Area X Reduces Song VariabilityFigure 2. Tissue measurements of DA biomarkers. (A) Immunoblot (40 lg protein/lane) from Area X, VSP, nidopallium (N), and mouse basalganglia (MBG) lysates. Signals are at the expected molecular weights (kD) and show the expected reduction in TH signal within nidopallium. (B)Immunoblot (15 lg protein/lane) from Area X and VSP lysates. Compared to vehicle (lane 1: 2.82, normalized protein levels), TH signalappeared reduced in Area X following both 0.6 lg (lane 2: 1.75) and 1.2 lg (lane 3: 1.03) doses of 6-OHDA, with more substantial reductionat the higher dose. TH signals in VSP exhibited less change as expected given the targeted injection to Area X (lanes 4–6; vehicle: 1.31; 0.6 lg:1.06, 1.2 lg: 0.93). (C) Decreased TH immunostaining in Area X following 6-OHDA injection. Photomicrographs show double-labeling for THpositive fibers in green and NeuN, a neuronal marker, in red. The star indicates the striato-pallidal border – beyond this border, the nidopalliumlacks the density of TH fibers. Arrowheads outline Area X (top); rectangle highlights inset shown below at higher magnification. There arefewer TH fibers (green) in the 6-OHDA injected Area X compared to vehicle-injected but the density of NeuN staining (red) indicates that AreaX neurons are preserved.in Area X that is visible to the naked eye. Within a givenbird, TH immunostaining was compared between thevehicle-injected versus 6-OHDA injected side. The tissuewas double-labeled with TH and the neuronal markerNeuN to confirm that Area X neurons were preserveddespite poisoning TH nerve terminals.Tissue sections were processed as follows: Hydrophobicborders were drawn on the slides, using a pap pen(ImmEdge, Vector Labs) followed by 3 9 5 min washesin TBS with 0.3% Triton X (Tx). To block non-specificantibody binding, the tissue was then incubated for 1 h atroom temperature with 5% goat serum in TBS/0.3% Txthen 3 9 5 min washes in 1% goat serum in TBS/0.3%Tx were performed. Primary antibodies to TH (Milliporerabbit 1:500), NeuN (Millipore #MAB377, mouse 1:500)were incubated in a solution of 1% goat serum in TBS/0.3% Tx overnight at 4 C. A “no primary antibody” control was included. The next day, 5 9 5 min washes inTBS/0.3% Tx were performed and sections were incubated for 4 h at room temperature in fluorescentlylabeled secondary antibodies (Molecular Probes/Life Technologies, 1:1000, goat anti-rabbit 488 #A11034; goat antimouse 546 #A11031). Following incubation, 5 9 5 minwashes were performed in TBS with filtered TBS used inthe last two washes. Slides were then coverslipped in ProLong Anti-Fade Gold mounting medium (MolecularProbes, #P36930), viewed on a confocal microscope (ZeissLSM 510) using Zeiss LSM software and analyzed, usingAdobe Photoshop. In Adobe Photoshop, mean intensityvalues for TH fiber staining were obtained by measuringthe same size rectangular area within Area X of bothhemispheres. Mean values for 6-OHDA were then dividedby vehicle values to obtain a percentage of TH fiber loss.ResultsValidation of DA biomarkersA polyclonal antibody made against TH (498 aa; GenBank: AAA42258.1) from rat pheochromocytoma wasused for detection. This antibody detects TH depletion inrat basal ganglia following 6-OHDA injection into themedial forebrain bundle (Ciucci et al. 2013). The immunizing peptide shares 76% identity to the predicted 491amino acids in zebra finch TH ( 55 kD; GenBank:XP 002198967). In immunoblots, a robust signal wasobserved at similar molecular weights across multiplebasal ganglia subregions in finch and mouse tissues(Fig. 2A). Signals were substantially reduced in the finchnidopallium, consistent with the reduced dopaminergicinnervation to this area relative to the basal ganglia inintact birds (Gale and Perkel 2005). A polyclonal antibodyagainst DARPP-32, previously used in zebra finches(Murugan et al. 2013), detects protein signal at theexpected molecular weight in region-specific areas of bothspecies ( 32 kD; Fig. 2A).Immunoblots revealed that bilateral injection of either0.6 lg or 1.2 lg of 6-OHDA into Area X reduced THlevels, with a more pronounced effect at the 1.2 lg dose(Fig. 2B). Additionally, fluorescent immunohistochemistrywas conducted on fixed coronal tissue sections from birdsreceiving a unilateral dose of 6-OHDA injected in Area Xof one hemisphere and vehicle in the other. In a representative section, intact, densely packed TH positive fiberswere detected throughout Area X in the vehicle-injectedhemisphere compared with decreased TH fiber staining(by 30%) in Area X in the 6-OHDA injected hemisphere(Fig. 2C). NeuN staining confirmed that only afferentfibers were lost as the neuronal cell bodies were still present in the 6-OHDA injected Area X (Fig. 2C).6-OHDA administration into Area X reduceslevels of TH but not DARPP-32 proteinA bilateral injection of 1.2 lg of 6-OHDA into Area Xsignificantly reduced TH signal relative to signals in vehicle-injected birds (Fig. 3A and B; n 4/group; mean SE: vehicle 1.47 0.19 vs. 6-OHDA 0.61 0.21; resampling mean difference P 0.006). In the outlying VSPfrom these same birds, no such reduction was observed(Fig. 3C and D, mean SE: vehicle 1.63 0.22 vs.6-OHDA 1.90 0.14; P 0.22) indicating that the neu-ª 2015 The Authors. 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J. E. Miller et al.6-OHDA Injection into Area X Reduces Song VariabilityArea XVehicleB6-OHDA75 kDTH50 kD37 kDArea X2.00GAPDHDARPP-32VSPD6-OHDA75 kDTH50 kD3
digitized (PreSonus Firepod/Audiobox: 44.1 kHz sampling rate/24 bit depth). Recordings were managed using Sound Analysis Pro (SAP) (Tchernichovski et al. 2000). The song was hand-segmented following Miller et al. (2010). Motifs were identified as a repeated order of mul-tiple syllables, excluding introductory notes and unlearned calls.
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32 Bageshree (registered) Bade Ghulam Ali Khan (concert Vocal mp3 33 Bageshree (registered) Hari Prasad Chaurasia ektal Bansuri / Flute mp3 34 Bageshree (registered) Kishori Amonkar Vocal mp3 35 Bageshree (registered) Rajab Ali Khan Vocal mp3 36 Bageshree (registered) Sya Ram Tiwari Vocal mp3 37 Bageshree 1 Hirabai Barodekar Vocal mp3 38 .
music (CCM) vocal pedagogy through the experiences of two vocal pedagogy teachers, the other in the USA and the other in Finland. The aim of this study has been to find out how the discipline presently looks from a vocal pedagogy teacher's viewpoint, what has the process of building higher education CCM vocal pedagogy courses been
(Nodes)A lump formed by an aggregate of cells on the vocal fold. Miller, 313 . Ossification. The hardening of tissue into a bony substance. Passaggio. Vocal register pivotal point (as in primo passaggio, secondo passaggio) Pedagogy (Vocal Pedagogy, Acoustic Vocal Pedagogy) Vocal pedagogy is the study of the art and science of voice instruction.
