Proc. Natl. Acad. Sci. USAVol. 92, pp. 5139-5143, May 1995Neurobiology

Feedback circuitry within a song-learning pathway(birdsong/brain/domains/myotopy/zebra finch)

G. EDWARD VATES AND FERNANDO NOTTEBOHMLaboratory of Animal Behavior, The Rockefeller University, New York, NY 10021

Contributed by Fernando Nottebohm, March 10, 1995

ABSTRACT The song system of birds consists of severalneural pathways. One of these, the anterior forebrain pathway,is necessary for the acquisition but not for the production oflearned song in zebra finches. It has been shown that theanterior forebrain pathway sequentially connects the follow-ing nuclei: the high vocal center, area X of lobus parolfacto-rius, the medial portion of the dorsolateral thalamic nucleus,the lateral magnocellular nucleus of anterior neostriatum(IMAN), and the robust nucleus ofthe archistriatum (RA).Wenow show in zebra finches (Taeniopygia guttata) that IMANcells that project to RA also project to area X, forming afeedback loop within the anterior forebrain pathway. Theaxonal endings of the IMAN projection into area X formcohesive and distinct domains. Small injections of tracer insubregions of area X backfill a spatially restricted subset ofcells in IMAN, that, in turn, send projections to RA that arearranged in horizontal layers, which may correspond to thefunctional representation ofvocal tract muscles demonstratedby others. We infer from our data that there is a myotopicrepresentation throughout the anterior forebrain pathway. Inaddition, we suggest that the parcellation of area X intosmaller domains by the projection from IMAN highlights afunctional architecture within X, which might correspond tounits of motor control, to the representation of acousticfeatures of song, or both.

Songbirds learn their song by reference to auditory informa-tion (1-3). A special set of nuclei and pathways governs thisprocess (Fig. 1). The main motor pathway goes from HVC toRA, which, in turn, innervates mesencephalic and medullarynuclei involved in phonation (4-8). HVC also projects to RAby a second pathway that includes nuclei in the rostral telen-cephalon. This other circuit, dubbed the anterior forebrainpathway, has previously been shown to connect sequentiallyHVC, area X of lobus parolfactorius, DLM, IMAN, and RA(9-11). In zebra finches, this anterior forebrain pathway isnecessary for song acquisition during development but not forsong production in adulthood (12-15). All of the nucleimentioned above respond to auditory stimuli, and in adults allrespond maximally to the bird's own song (6, 11, 16-19). It isassumed that auditory information is fed into the direct andanterior forebrain pathways from HVC (16, 17, 20). Since theanterior forebrain pathway is essential for song learning, whichinvolves matching vocal output to learned auditory models, wewere interested in more clearly identifying the anatomicalrelations between its component nuclei. Such informationcould provide clues as to how the anterior forebrain pathwayorganizes auditory or motor functions that serve to guide vocallearning.The present report demonstrates a previously undescribed

projection from IMAN to area X. This projection originates inthe same IMAN cells that project to RA. In addition, IMANhas other interesting features: (i) neurons in subregions of

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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FIG. 1. Schematic of the major nuclei of the song circuit. The nucleiconnected by solid arrows, the high vocal center (HVC), the robustnucleus of the archistriatum (RA), the dorsomedial nucleus of theintercollicular complex (DM), and the tracheosyringeal portion of thehypoglossal nucleus (nXIIts), are part of the direct descending motorpathway for song. The anterior forebrain pathway (stippled arrows)connects HVC to area X, the medial portion of the dorsolateralthalamic nucleus (DLM), and the lateral magnocellular nucleus ofanterior neostriatum (lMAN) before continuing to RA. A white arrowmarks the newly discovered connection reported in this paper.

IMAN project to horizontal layers in RA that may correspondto the myotopic representation described by Vicario (21), (ii)neurons from the same IMAN subregions also project to areaX in a topographic fashion, and (iii) the terminals of individualIMAN neurons in area X fall within discrete, cohesive do-mains. "We suggest that, as is the case for projections fromIMAN to RA, so too the connections that link area X to DLM,DLM to IMAN, and IMAN to area X are topographicallyorganized and that therefore each of these nuclei contains amyotopic representation." We propose that the domains inarea X identified by the projections from IMAN representfunctional processing units whose study will allow greaterunderstanding of the role area X plays in song learning.

MATERIALS AND METHODSExperiments were conducted with 16 adult male zebra finches(Taeniopygia guttata, >120 days old) obtained from our ownbreeding colonies at the Rockefeller University Field Re-search Center (Millbrook, NY).

Abbreviations: DLM, medial nucleus of the dorsolateral thalamus;HVC, high vocal center; IMAN, lateral magnocellular nucleus of theanterior neostriatum; RA, robust nucleus of the archistriatum; FLG,fluorogold; BDA, biotinylated dextran amine; HVC, high vocal center.

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Injections of Neuronal Tract Tracers. Ten birds receivedinjections of biotinylated dextran amine [BDA; 10% in phos-phate-buffered saline (PBS); Mr 3000; Molecular Probes]targeted to RA bilaterally (four birds), RA unilaterally (onebird), area X bilaterally (four birds), and area X unilaterally(one bird). Additionally, two birds received bilateral injectionsof fluorogold (FLG, 2% in saline) into RA as well as bilateralinjections of rhodamine-linked latex microspheres (beads;Lumafluor, New York) into area X, and two birds receivedbilateral injections of FLG into RA and bilateral injections oftetramethylrhodamine-linked dextran amine (RDA; 10% inPBS; Mr 3000; Molecular Probes) into area X.For all surgery, anesthesia was induced with Nembutal

(Abbott) and maintained with Metofane (Pitman-Moore,Washington Crossing, NJ). Each bird was placed in a ster-eotaxic apparatus and the skin over the skull was opened. RA,area X, and IMAN were injected using glass micropipettes(o.d., 10-30 p,m) at previously determined stereotaxic coor-dinates based on modifications of a canary atlas (22). BDA andRDA were injected iontophoretically [3- to 5-,uA current,10-20 min, 7 s on/off; Midguard Transkinetics (Canton MA)current source]. FLG and beads were pressure injected (10-20nl) using a hydraulic microinjection system (Narishige, Tokyo).Penetration tracks were angled as necessary to prevent leakageof tracers to structures of interest as described (14, 23), andsmall injections were used to further limit spread. Four daysafter injection, birds were killed under deep Nembutal anes-thesia. Fixation was by intracardiac perfusion with 10 ml ofsaline, followed by 50 ml of 4% paraformaldehyde in 0.1 Mphosphate buffer (pH 7.4). Brains were removed and incu-bated overnight in the same fixative at 4°C.

Histology. For brains injected with BDA, each brain hemi-sphere was cut serially into 50-,um sagittal sections on avibratome (Lancer, Longwood, FL) and collected in PBS.Every other section was then allowed to react to visualize BDAby first incubating in 5% skim milk/0.3% Triton X-100 in PBS,followed by avidin-biotin complex solution (ABC; VectastainElite Kit, Vector Laboratories). After several rinses in PBS,BDA-filled cells were revealed by incubation in 0.03% 3,3'-diaminobenzidine (DAB)/0.15% nickel ammonium sulfate/0.001% H202 in PBS. Sections were subsequently rinsed inPBS, mounted onto chromalum slides, air dried overnight,delipidized in xylene, and coverslipped with Krystalon. Alter-nate sections were not allowed to react in ABC/DAB but,instead, were mounted immediately and counterstained with0.5% cresyl violet acetate.For brains injected with fluorescent tracers, each brain

hemisphere was soaked in increasing concentrations of sucrosein 4% paraformaldehyde until equilibrated in 30% sucrose in4% paraformaldehyde. Each hemisphere was then cut seriallyinto 50-Am sagittal sections on a sliding freezing microtome(American Optical) and collected in PBS. Each section was

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Microscopy. Sections of interest were mapped using acomputer-yoked microscope (24), which allows counting oflabeled cells within different nuclei in each section, as well asmeasuring cross-sectional areas of different regions of interestused for volume calculations.

RESULTSInjections of BDA limited to RA backfilled many neuronswithin HVC and IMAN (Fig. 2); we confirmed that theseconnections were ipsilateral in an animal that received aninjection ofBDA into RA on one side only (not shown). BDAtravels in anterograde and retrograde directions and, in manyinstances, provides backfills of neurons that appear complete,similar to Golgi material (25, 26). There was clear labeling notonly of dendritic features but also of axons, and their collat-erals could be followed for long distances. As reported byothers (27), we observed no cells in HVC that extended axoncollaterals to other targets. In contrast, many of the cellsbackfilled in IMAN sent axon collaterals that could be followedthrough serial sections to their presumed terminals in area X(Fig. 3). These results indicated that many cells in IMAN thatproject to RA simultaneously project to area X.

Because this finding contradicted the results of many otherreports (9-11), the pattern of connectivity was confirmed intwo ways. (i) Injections of BDA directly into IMAN revealedanterograde projections to area X (not shown) as well as itsknown projection to RA. Interestingly, the endings of projec-tions from IMAN to RA appeared to project preferentially tothe ventral two-thirds of RA, which innervates syringeal motorneurons (21). Fibers found in the dorsal one-third ofRA wereless densely packed, and most appeared to be fibers of passage.This suggests that IMAN does not have a strong projection tothe dorsal subregion of RA, which innervates mesencephalicand medullary targets involved in control of respiratory mus-cles (21, 28-30). (ii) Injections of BDA and RDA into area Xbackfilled neurons in IMAN (Fig. 4) as well as the expectedcells in HVC; however, the distribution of backfilled cells inthese two nuclei was very different and will be described later.We confirmed that the connection from IMAN to area X wasipsilateral in an animal that received an injection of BDA intoarea X on one side only (not shown).To confirm that single IMAN cells projected to area X and

RA, and to rule out the possibility that cells with a similar dualprojection occurred in HVC, we injected FLG into RA andrhodamine beads or RDA into area X. When we did this wesaw many individual neurons in IMAN that were doublylabeled with FLG from RA and with one of the otherfluorescent tracers from area X (Fig. 5). We saw no double-

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FIG. 2. Photomicrographs of backfilled neurons in HVC (a) and 1MAN (b) in sagittal section after injection of BDA in RA. Arrowheadsin a mark the ventral border of HVC and in b mark the boundaries of 1MAN. Dorsal is up, and anterior is to the right. (Bars: a, 200 ,um; b,100 I&m.)

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FIG. 3. (a) Photomicrograph of Nissl-stained sagittal section of zebra finch brain showing IMAN (arrowheads) and area X (dotted line). (b)Same-magnification view of an alternate section showing backfilled neurons in IMAN after injection ofBDA in RA. Axon collaterals from backfilledIMAN neurons project into area X, forming cohesive terminal domains (black arrows). (c) Dark-field photomicrograph of axon collaterals leavingIMAN for RA (open arrow) and area X (solid arrow). (d) Photomicrograph of the two terminal domains seen in b. (e) High-magnification viewof the right terminal domain in d. In all photomicrographs, dorsal is up, and anterior is to the right. (Bars: a, 500 ,m; c, 200 Jim; d, 100 ,Lm; e,50 ,im.)labeled cells in HVC, which coincides with previous observa-tions (27). Our methods do not allow us to conclude that all ofthe 1MAN cells that project to RA also project to area X,because a complete fill of area X with retrograde tracer wouldinevitably leak into 1MAN. However, we took advantage of thefact that the distribution of cells in 1MAN backfilled from asubregion of area X was limited, in turn, to subregions of1MAN (see below). We defined a subregion of 1MAN forquantitative study by drawing a border around collections of1MAN neurons backfilled from area X. In that subregion, wecounted the number of neurons labeled with FLG from RA,the number of neurons labeled with BDA or beads from areaX, and the number of double-labeled cells. These measure-ments showed that 63% of 1MAN neurons that projected toRA also sent collaterals to area X (n = 3 double-injectedhemispheres). Only 5% of cells backfilled from area X did notdemonstrate any FLG backfill from RA. Because we knowfrom past experience that FLG is an effective retrograde tracer(15, 23), but cannot be sure that any of the other tracers weused fill all of the cells that project to a given target, our resultsprobably underestimate the true number of double-projectingcells.As mentioned above, we observed differences in the distri-

bution of backfilled cells in HVC and 1MAN after injections ofBDA and fluorescent tracers into area X. Whereas backfilledcells in HVC were distributed throughout the nucleus regard-less of the site of injection in area X, backfilled neurons in1MAN were restricted to a particular sector, which dependedon the site of injection. For example, injections in anterior,posterior, or ventral area X backfilled neurons in the anterior,

posterior, or ventral portions of 1MAN, respectively (Fig. 4).This indicates that the projection from 1MAN to area X isarranged in a topographic manner. We did not investigatewhether there was any further topography in the mediolateralplane.These same small injections of BDA into area X also

revealed spatially restricted terminal fields within DLM, theonly known target of area X (not shown). Thus, area X seemsto receive a topographically organized projection from IMANbut not from HVC and then projects in a topographic fashionto DLM. Taken together with preliminary observations thatdemonstrate a topographic mapping of connections fromDLM to IMAN (31), it appears that the entire anteriorforebrain pathway is topographically connected.

Detailed study of the projections of IMAN neurons back-filled from area X demonstrated further topography in theirprojection to RA (Fig. 6). Neurons in the posterior portion ofIMAN sent axons that terminated in a roughly horizontal slabthrough the middle third of RA. In contrast, neurons in theanterior portion ofIMAN sent axons that terminated in a bandin the ventral one-third of RA. As mentioned above, injectionsof BDA directly into IMAN resulted in terminal labeling thatwas dense in the ventral two-thirds of RA but sparse in thedorsal one-third. Upon comparison with the results of Vicario(21, 28, 29), our observations suggest that neurons in differentregions of IMAN project to portions of RA that are dedicatedto control of specific syringeal muscles, but do not project asdensely to portions ofRA that coordinate respiratory functionwith singing. It should be noted, however, that we do not yethave injections involving IMAN in all of its mediolateral extent

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FIG. 4. (a and b) Photomicrographs of small BDA injections inposterior or anterior area X, respectively. (c) Photomicrograph ofsmall RDA injection in ventral area X. (d-f) Photomicrographsshowing location of cells in IMAN backfilled from injections seen ina-c, respectively. Backfills reveal that different parts of IMAN projectto corresponding sectors of area X. The arrow in e points to the smallnumber of cells backfilled from the small injection site. (Bars: a, 300,um; d, 150 ,um.)

and cannot rule out the possibility that the lateral-most regionsof IMAN may innervate the dorsal subregion of RA morecompletely.The injections of BDA into RA that revealed the projection

from IMAN to area X also showed that the terminals in areaX had an unusual morphology. Because the iontophoreticinjections ofBDA into RA were small, involving 25-40% of itstotal volume in each case, the number of backfilled cells inIMAN did not include every cell that projected to RA, and the

FIG. 5. High-power view ofIMAN neurons that demonstrates FLGlabeling (a) and RDA labeling (b) resulting from an injection of FLGinto RA and RDA in area X. (Bar = 150 jam.)

number of these cells that were densely backfilled was evensmaller. Thus, this technique revealed only a subpopulation ofthe terminals sent from IMAN to area X. This partial repre-sentation, however, helped us see that the terminals sent byIMAN to areaX formed discrete and compact fields of endings(Fig. 3 d and e). Examples of terminal domains, chosen fromthe center of the nucleus, appeared roughly spherical and hada mean diameter of 178 ± 24 Am (n = 18 from three animals).These domains appeared to originate from one or more axonalfibers and consisted of convoluted and feathery endings withfew, if any, varicosities. Based on measurements of the volumeof area X from cresyl-stained alternate sections (mean volume= 1.78 ± 0.13 mm3, n = three birds), we estimate that =600of these domains can fit within an average volume for area Xif there is no overlap. However, this may represent an under-estimate for a number of reasons. (i) Terminal fields at theedge of areaX appear to occupy a significantly smaller volume.(ii) We chose robust examples of terminal domains that wereeasily mapped and may have excluded smaller domains thatmight not stand out as dramatically against a complex back-ground of passing fibers. (iii) If our assumption that thespheres are mutually exclusive is not true, then many moredomains could fit within area X. Regardless of the number, itis clear from these findings that the projection from IMANdefines a specifically organized architecture within area X.

DISCUSSIONThis report documents a previously undescribed connection inthe anterior forebrain pathway of songbirds and providesevidence of topographic organization within this pathway thatrelates to the functional architecture of RA. By injecting atracer into RA that is carried retrogradely and anterogradely(BDA), we were able to backfill cells in IMAN that were thenseen to project into area X. This projection from IMAN to areaX is topographic, as demonstrated by small injections of BDA

FIG. 6. (Left) Lateral-to-medial series of three cameral lucida maps of sections through RA, showing the distribution of terminals in RA comingfrom lMAN neurons backfilled by the injection of BDA into area X shown in Fig. 4a. (Right) Similar series of camera lucida maps of terminalsin sections ofRA resulting from the injection ofBDA into area X shown in Fig. 4b. (Left Inset) Photomicrograph corresponding to the most medialdrawing. (Right Inset) Photomicrograph corresponding to the middle drawing.

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into area X; these same injections then revealed the horizon-tally layered projections from IMAN to RA that may corre-spond to the functional organization of the syringeal controlareas in RA discovered by Vicario (21). The injections intoarea X also showed topographic connections from area X toDLM. Since Johnson et at (31) have shown that DLM projectstopographically onto IMAN, we suggest that the same myo-topic pattern seen in RA is also present in IMAN and in eachof the nuclei of the anterior forebrain pathway.Our anatomical data suggest that IMAN provides some kind

of feedback to area X. In addition, the distinctive appearanceof lMAN's terminals in area X is particularly intriguing. Itsuggests that area X is parceled into a number of differentfunctional units. These domains in area X may correspond toprocessing "modules." If the domains revealed by lMAN'sprojections correspond to separate processing units, then theirnumber may be surprisingly limited. Since the number of areaX cells that project to DLM represents