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Introduction

Vocal learning is the ability to imitate vocalization from others, and is a critical behavioral substrate for spoken human language. As such, vocal learning is a rare trait found to date in at least three distantly related groups of mammals (humans, bats, and cetaceans [1]; also see recent studies including seals [1], [2] and elephants [3]), and in three distantly related groups of birds (songbirds, hummingbirds, and parrots [4]; Fig. 1A). Among the birds, vocalizing-driven immediate early gene (IEG) expression experiments revealed that each vocal learning group possesses seven comparable cerebral (i.e. telencephalic) song nuclei, also called vocal nuclei (Fig. 1B) [5]–[7]. Three of the nuclei make up part of an anterior vocal pathway (Fig. 1B, red) that in songbirds is necessary for vocal learning [8]–[10]. The other four nuclei form a posterior vocal pathway (Fig. 1B, yellow) of which the songbird HVC and RA are necessary for the production of the learned vocalizations [10]–[12] (abbreviations in Table 1). None of these cerebral vocal nuclei have been found to date in vocal non-learners, such as in the suboscine songbirds closely related to songbirds [13], [14], the interrelated doves [15], [16], and the distantly related galliforms ([17], Fig. 1B). Yet, both vocal learners and non-learners have brainstem vocal nuclei DM and nXIIts (Fig. 1B) that are responsible for production of innate vocalizations [7], [18], and an auditory cerebral pathway that is responsible for processing species-specific vocalizations and auditory learning ([19]–[21], Fig. 1B, blue). Therefore, according to the dominant hypothesis [4], [14], within the past 65 million years 3 out of 23 avian orders independently evolved seven similar cerebral vocal nuclei for a complex behavior ([7], Fig. 1A, red dots). The reason for these remarkable similarities had remained mysterious, but they suggest that the evolution of brain structures for vocal learning is under strong genetic or epigenetic constraints. Here we conducted a series of experiments that, in addition to identifying the motor control circuit of the avian brain, points towards a possible genetic constraint: a pre-existing motor system that in birds consists of at least seven cerebral areas active during the production of limb and body movements. Of the multiple hypotheses proposed for the evolution of vocal learning [14], [22], [23], including for spoken language, our findings support those that suggest a motor origin [10], [24], [25].

Figure 1. Phylogenetic and brain relationships of avian vocal learners.

A. One view of the phylogenetic relationships of living birds [129]. Vocal learners are highlighted in red. Red dots: possible independent gains of vocal learning; green dots: alternatively, possible independent losses. B. Semi-3D view of seven cerebral vocal nuclei (red and yellow) found in vocal learners and of auditory areas (blue) found in all birds. Red-labeled vocal nuclei and white arrows: anterior vocal pathway. Yellow-labeled vocal nuclei and black arrows: posterior vocal pathway. Only a few connections in hummingbirds are known and that of songbird MO is not known. Based on serial sections of singing-driven IEG expression in this study, we see that NIf and Av are adjacent at lateral levels. Anterior is right, dorsal is up. Scale bars, 1 mm. Figure modified from Jarvis et al. (2000) [7] and Jarvis (2004) [10], with connectivity reviewed therein.

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Table 1. Terminology of comparable regions across avian species studied

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Results

In experiments that identified night vision brain areas in migratory songbirds [26], [27], we performed a series of control experiments that led to the identification of brain areas associated with movement behavior that we report here. Unexpectedly, the areas of robust movement-associated activation were closest to the vocal nuclei, and thus we investigated this activation further in a non-migratory songbird, the zebra finch (Taeniopygia guttata), for which the vocal system has been studied in detail. Once we established these areas as movement-associated and adjacent to vocal nuclei in songbirds (Part I of this report), we next tested whether other vocal learning birds (Part II) and vocal non-learning birds (Part III) had similar properties to address implications on the evolution of vocal learning. To perform the experiments, we compared groups of birds that repeatedly performed specific movements for 30–60 min with control groups that either sat still but awake or were in other experimental conditions. The movement behaviors were wing whirring, flights, hopping, or walking. We sectioned the brains and performed in-situ hybridizations for the activity-dependent IEG transcription factors ZENK and c-fos. The mRNA expression of these genes is sensitive to increased activity in neurons [28]. We processed adjacent sections for expression of the AMPA glutamate receptor subunit GluR1 or the transcription factor FoxP1, because we found that these anatomical markers were critical for identifying the different cerebral subdivisions in each species, particularly the mesopallium [16], [29], that were otherwise prone to identification errors with Nissl staining (see anatomy section in methods for a detailed explanation). The results represent hybridizations to over 5,800 serially cut brain sections of entire hemispheres from five species. In species where the brain regions, such as vocal nuclei, are thought to have evolved independently, we used different names following previous designations [7], [30] and the new avian brain nomenclature [29], [31]. Based on their functions, we refer to the ‘posterior vocal pathway’ as a ‘vocal motor pathway’ and the ‘anterior vocal pathway’ as a ‘vocal learning pathway’. However, when we refer to vocal learning nuclei or system, we mean all cerebral vocal/song nuclei in vocal learners, as these nuclei are associated with the presence of vocal learning and also have motor related-neural firing (in songbirds) and/or IEG expression [5]–[7], [32]–[34]. Figure 2 shows examples of the most reduced movement-associated and singing-driven patterns in songbirds we have obtained. Table 1 lists all anatomical regions studied and their relative similarities across species.

Figure 2. IEG expression patterns in brain sections from moving versus singing zebra finches.

A. Example of the most restricted movement-associated expression pattern obtained in this study. A mid-sagittal section from a deafened bird that was hopping in the dark. B. Comparable section from a bird that was singing while movingly relatively little. ZENK expression is shown in (A); c-fos is shown in (B) due to its high contrast in vocal nuclei. White: IEG expression; red: cresyl violet staining. Six (HVC, NIf, Av, Area X, MAN, and MO) of the seven vocal nuclei can be seen in these sections, which are adjacent to five movement-associated areas (PLN, PLMV, ASt, AN, AMV); movement-associated areas laterally adjacent to the HVC and RA are not in this section. AMD and AH are known somatosensory areas. Anterior is right, dorsal is up. Scale bar, 2 mm.

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Part I. Movement-Associated Brain Areas in Songbirds

Migratory restlessness: wing whirring and flights.

Migratory restlessness is a highly stereotyped and repetitive behavior performed by migratory songbirds at night in dim light, during their migratory season [35], [36]. We found that under dim light in our cylindrical monitoring apparatus (Supporting Fig. S1A), migratory songbirds performed migratory restlessness behavior consisting mainly of rapid wing whirring while perched, but also head scans and some hops, in a preferred direction corresponding to the migratory orientation of their free-flying conspecifics [26], [37]. Using infrared video, we quantified wing whirring in garden warblers (Sylvia borin) in dim light and flights in day light for 45–60 min. Relative to birds that sat still (Fig 3Aa-d), those that performed flights (Fig. 3Ae,f) or wing whirring (Fig. 3Ag,h) both had high levels of induced ZENK gene expression in the medial part of the anterior cerebrum centered around the anterior vocal nuclei and in areas lateral to the posterior vocal nuclei. The anterior activated areas included the medial part of the anterior striatum (ASt) centered around the striatal vocal nucleus Area X, the anterior nidopallium (AN) centered around the magnocellular vocal nucleus of the anterior nidopallium (MAN, both lateral and medial parts), and the anterior ventral mesopallium (AMV) centered around the mesopallium oval (MO) vocal nucleus; we note here that the MO vocal nucleus is situated in the ventral mesopallium (MV), as revealed by comparisons of GluR1 with ZENK expression (Fig. S2A; also seen below for zebra finches and FoxP1 expression). The caudal limits of the anterior activated areas (ASt, AN, and AMV) in birds that made flights formed a semi-linear functional boundary across the respective brain subdivisions (St, N, and MV; Fig. 3Ae). Other activated areas were the medial part of the dorsal mesopallium (MD) and adjacent medial hyperpallium (H; Fig. 3Ae, 3B). The posterior activated areas included the dorsal lateral nidopallium (DLN) adjacent to the HVC vocal nucleus and the lateral intermediate arcopallium (LAI) adjacent to the robust arcopallium (RA) vocal nucleus (Fig. 3Af,h, 3B; Fig. S2B shows higher power images). Expression was also high in the posterior lateral nidopallium (PLN) and adjacent posterior-lateral ventral mesopallium (PLMV; Fig. 3Af,h) lateral to the two other posterior vocal nuclei-nucleus interface of the nidopallium (NIf) and avalanche (Av) of the ventral mesopallium. However, it was difficult to determine whether NIf and Av were immediately adjacent to the vocal nuclei in garden warblers, as these vocal nuclei are small and more easily identified by singing-driven gene expression (see experiments below with zebra finches).

Figure 3. Movement-induced ZENK expression in garden warblers.

A. Darkfield images of medial (top) and lateral (bottom row) sagittal sections from birds sitting relatively still in room day light (a, b) or dim night light (c, d) and birds making mostly flights in day light (e, f) or wing whirring in dim light (g, h). The anatomical profiles to the right show the extent of the movement-induced (red) and visual-induced (blue) gene expression areas. Note that the PLN and PLMV areas differ slightly in shape between garden warblers (panel Af,h) and zebra finches (Fig. 2A), due to differences in the shapes of the N and MV cerebral subdivisions and that the warbler sections are more lateral. Anterior is right, dorsal is up. Scale bar, 2 mm. B. Quantification of ZENK expression levels in different brain areas from the four groups of warblers. Anterior and posterior areas were grouped according to their relative location to vocal nuclei. * = p<0.05, one-way ANOVA followed by Holm-Sidak multi-comparison test, comparing moving groups with still groups for each light condition; each movement group showed significant differences with each still group. # = p<0.001 for an increase in Cluster N in dim versus day light groups, whether still or moving. Error bars, S.E.M. C. Correlation between the amount of wing beats and ZENK expression levels shown in exponential (top row) and double natural logarithmic (bottom row) graphs for example areas. Each dot represents the value of one bird. D. Statistical analyses: (a) one-way ANOVA followed by Holm-Sidak all-pairwise multi-comparison test for the brain areas in (B); (b) exponential regression stats (examples in C, top row); (c) linear regression stats on double-logarithmic transformation of the data (C, bottom row). Red text are significant differences (p<0.05); n.s. = not significant.

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Although ZENK expression can be found in other brains areas of individual animals or of a group, activation in the brain areas adjacent to the vocal nuclei (3 anterior, 4 posterior) as well as throughout medial MD and medial H occurred whether the birds performed wing movements in day or dim light (Fig. 3B; statistics in 3Da, 3^rd & 4^th rows). In control areas such as the hippocampus and the higher auditory area NCM, there were no increases in expression that were exclusive to the movement groups (Fig. 3B, 3Da). We also measured two regions as background controls, the entopallium (E, a primary visual region) and the globus pallidus (GP) known not to express high levels of ZENK [6], [38], and found no significant differences among groups (Fig. 3B, 3Da).

Outside the cerebrum, we noted distinct activation in the granule cell layer of specific cerebellum lobules (Fig. 4A–C). Birds that performed flights and were generally active had ZENK activation distributed across lobules I-X (Figs. 3B and 4B) albeit with higher levels in lobule VIab (p<0.01, relative to IXcd, paired t-test), whereas birds that performed wing whirring while perched had high activation specific to lobules I-VI (Fig. 4C, p<0.012, VIab versus IXcd, paired t-test).

Figure 4. Movement-induced ZENK expression in the cerebellum.

Example sagittal sections of: A. Garden warbler sitting still. B. Garden warbler making flights and other movements in day light. C. Garden warbler performing wing whirring in dim light. D. Zebra finch sitting still. E. Zebra finch flying and hopping around the perimeter of the cylinder cage. F. Zebra finch hopping in a rotating wheel. G. Budgerigar hopping in the wheel. H. Anna's hummingbird hovering. I. Ring dove walking on a treadmill. Birds in G and I were deaf; F, G, and I were in the dark. Anterior is right, dorsal is up. Scale bar, 1 mm.

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Proportionality.

In all vocal learning birds, IEG induction levels in vocal nuclei is linearly proportional to the amount of songs produced per 30–60 min and this has been one test to support the conclusion that singing-associated gene expression is motor-driven [5]–[7], [33]. To test for this property in the identified areas, we performed regression analyses on the amount of gene expression with the number of wing beats (counted semi-automatically, both during wing whirring and flights). Similar to the vocal nuclei, the amount of IEG expression in the adjacent anterior and posterior areas, and in the cerebellum, was proportional to the amount of wing beats and flights performed (Fig. 3C, top panels and 3Db). Interestingly, the relationship was not linear, but was best fitted by a saturating exponential curve (Fig. 3C top), similar to the relationship seen in the number of neurons that express ZENK protein in vocal nuclei when birds sing [39]. ZENK mRNA expression levels reached a maximum at ~6000 wing beats and then saturated, and may have even started to decrease (or perhaps habituate [40]) in some birds. Natural logarithmic transformations of the data were linear (Fig. 3C, bottom panels and 3Dc), confirming that the original relationship was exponential. In both exponential and logarithmic analyses, the correlations were stronger for the anterior areas and the cerebellum (Fig. 3Db,c). There were no correlations between wing beats or flights and ZENK expression for the hippocampus, NCM, or the E (Fig. 3Db,c). A significant but barely detectable correlation with the number of flights was seen in GP, but this is not surprising as the GP is generally known to be involved in motor control [29].

Hopping and walking.

We next analyzed brains of a non-migratory songbird, the zebra finch, which was placed in our cylindrical apparatus. In the day light condition, most zebra finches hopped and walked around the perimeter of the cage at a rapid pace. The ZENK expression pattern in these finches relative to sitting still controls (in dim light, Fig. 5Aa) was similar to that found in the flying day time group of garden warblers, except that the activation of the anterior areas (ASt, AN, and AMV) was more narrowly focused around the anterior vocal nuclei (Fig. 5Ab, 5B). Likewise, the caudal and rostral limits of high expression each lined up to form a strip of activation across the three brain subdivisions: St, N, and MV. Activation in the medial MD and adjacent medial H still spanned the caudal-to-rostral extent of their respective brain subdivisions (Fig. 5Ab). The cerebellum showed a gradient of high expression concentrated towards lobule VI (Fig. 4E), similar, but not identical to that seen in garden warblers that performed flights (Fig. 4B), and clearly increased relative to finches that sat still (Fig. 4D and 5B).

Figure 5. Movement-induced ZENK expression in zebra finches.

A. Sagittal sections of birds (a) sitting relatively still in dim light, (b) hopping around the perimeter of a cylindrical cage in day light, (c) hopping in a rotating wheel in the dark, (d) sitting still in the dark and hearing a bird hop in the rotating wheel, (e) sitting still in the dark and hearing playbacks of zebra finch song, and (f) hopping in the rotating wheel in the dark while deaf. The anatomical profile in the lower right highlights the extent of the movement-induced (red) and visual- and auditory-induced (blue) gene expression. Anterior is right, dorsal is up. Scale bar, 2 mm. B. Quantification of ZENK expression levels in 23 brain regions in 8 groups of male zebra finches. Except for a small difference in cerebellum lobule VI, there were no significant differences between sitting still dim light and dark animals; thus, they were treated as one control group for statistical analysis. * = p<0.05 to<0.0001, one-way ANOVA followed by Holm-Sidak multi-comparison test relative to combined values of sitting still animals in dim light+dark (n = 3–6/group). Lines underneath *s indicate brain areas with statistically significant increases exclusive to the moving groups. #s alone indicate values that approached significance by a few tenths of decimal in the ANOVA test. Error bars, S.E.M.

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In the above experiments, the movement behaviors were performed voluntarily. Although the birds performed repetitive stereotyped movements, there were still a variety of types across and within animals. To induce a more repetitive single-movement performance, we developed a motorized rotating plexiglass wheel orientated sideways (similar to a hamster wheel, Fig. S1B). When the wheel was rotated at a constant rate, in dim light, the zebra finches repeatedly hopped, with a minimum of other movements, to maintain their position at the bottom of the rotating wheel. The ZENK expression pattern in these zebra finches was similar to that in birds that voluntarily hopped around the cage (Fig. 5B), though more distinct (shown below in detail for other groups in the wheel) as expected due to mainly one type of movement performed. The activation in the cerebellum was more restricted to the anterior lobules (highest in VI) and in the posterior lobule IXcd (Fig. 4F), similar, although not identical to that seen in garden warblers that performed wing whirring behavior (Fig. 4C).

Visual versus movement activation.

In both garden warblers and zebra finches we noted differential activation in other brain areas that varied across groups (Fig. 3B, control regions). Some of these areas are parts of known visual pathways. They included: 1) the nidopallium and ventral mesopallium adjacent to the entopallium that we here call Ne and MVe (Fig. 3Af), which are part of the tectofugal visual pathway and that we previously demonstrated were activated by day-light vision [26]; 2) the posterior part of the medial hyperpallium (PH, also known as the visual Wulst) and the adjacent posterior dorsal mesopallium (PMD; Fig. 5Ab), which are part of the thalamofugal visual pathway [41]; and 3) cluster N, consisting of regions within the most posterior end of the hyperpallium and adjacent dorsal mesopallium (Fig. 3Ah) that we previously demonstrated was activated by dim-light, night-vision in migratory songbirds and implicated in light-mediated magnetic compass detection [26], [42]. We found that cluster N in garden warblers showed strong induced expression by dim light whether the birds sat still or performed migratory restlessness, with no detectable effect of movement (Fig. 3Ad,h, 3B, 3Da). Ne and MVe showed strong induced expression by day light relative to dim light (Fig. 3Ab,f, 3B, 3Da); however, Ne and MVe showed further increased expression in the moving relative to the respective day and dim light control sitting still birds (Fig. 3B, 3Da), with stronger correlations with the number of flights than with the number of wing beats (Fig. 3Db,c). Because flights cause optic flow more so than stationary wing whirring movement, we surmise that Ne, proposed to detect visual motion [43], as well as MVe, might detect optic flow as the birds move.

If visual areas are activated as a result of motion detection, then movement in complete darkness should eliminate this activation. To test this idea, we placed zebra finches in the rotating wheel in complete darkness and observed their movements using an infrared camera. They hopped similarly as the birds moving in dim light. We found that hopping in darkness eliminated the widespread induced expression in visual areas (PMD, PH, Ne and MVe; Fig. 5Ac, 5B; in-situ shown only for PMD and PH). In contrast, high levels of induced ZENK expression remained in the areas (ASt, AN, and AMV) surrounding the anterior vocal nuclei, the areas (DLN, LAI, PLN, and PLMV) laterally adjacent to the posterior vocal nuclei, known somatosensory areas of the anterior dorsal mesopallium (AMD) and adjacent anterior hyperpallium (AH), and a small strip of cells in the nidopallium (Nb) and ventral mesopallium (MVb) adjacent to the second somatosensory area basorostralis (B; Nb and MVb shown in Fig. 6C below, [44]); B like the E does not express high levels of ZENK [38]. High levels of induced ZENK expression also remained in the cerebellum of birds hopping in the dark (Fig. 5B). Thus, we conclude that the movement-associated gene expression in the cerebellum, regions of the two known somatosensory pathways, and the regions adjacent to the anterior and posterior vocal pathway nuclei is independent of visual input.

Figure 6. Serial sagittal brain sections of ZENK expression in male zebra finches.

A. Auditory areas: bird sitting still in the dark while hearing song. Although not mentioned in the main text, hearing-induced expression also occurs in caudal St and MLd, known auditory regions of the striatum and midbrain, respectively [45]. B. Auditory and vocal areas: bird hearing and singing alone in a sound box in the day light condition. C. Movement areas: bird hopping in the rotating wheel in the dark while deaf. D. FoxP1 expression from adjacent sections of the bird in (C). E. Corresponding anatomical drawings; red: areas with movement-induced expression; blue: areas with auditory- or visual-induced expression. First row are medial-most sections. Anterior is right, dorsal is up. Scale bar, 2 mm. Compare with frontal sections in figure S3.

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Auditory versus movement activation.

We noted that in some groups of moving birds there was increased ZENK expression in some of the known auditory areas surrounding the primary auditory field L2 (L2, like the E and B, does not express high levels of ZENK [45]). Such areas include fields L1, L3, and surround (called N-L2 here) and CM (called MV-L2 here), which particularly showed increased ZENK expression in garden warblers that performed wing whirring (Fig. 3Ag, 3B, 3Da) and in zebra finches that performed hopping in the rotating wheel (Fig. 5Ac, 5B), even though the wheel was in a sound isolation box with the motor mounted outside of it (Fig. S1B). N-L2 expression in garden warblers had a stronger correlation with the number of wing beats than with the number of flights (Fig 3Db,c). We surmise that the auditory pathway may be responding to self-produced wing whirring sounds and to hopping sounds on the wheel floor or mechanical sounds transmitted through the rotating wheel, similar to the IEG induction that occurs in N-L2 and other auditory areas when songbirds hear themselves sing [33]. But some reports noted that song playbacks alone can also result in increased ZENK expression in the anterior striatum surrounding Area X, where it has been suggested that the forebrain vocal pathway may be located within an auditory system [46], [47]. This idea was further supported by the facts that HVC sits dorsally adjacent to the HVC shelf and RA caudally adjacent to the RA cup, two known auditory pathway areas with hearing-induced IEG expression [23], [33], [45].

To assess the plausibility of these ideas, we individually placed zebra finches in complete darkness outside of the wheel next to a yoked control bird hopping inside the wheel, of which the former bird could hear the wheel rotate and the bird hop inside of it, but did not move himself and could not see the bird hop. These sitting still, but hearing stimulated birds showed high induced ZENK expression in the N-L2, MV-L2 and other auditory areas, but had no detectable increases in the 11 cerebral regions that include the areas surrounding the anterior vocal nuclei (ASt, AN, and AMV), lateral to the posterior vocal nuclei (DLN, LAI, PLN, and PLMV), and the somatosensory areas (Nb, AMD, AH; except for a small increase in MVb), or in the cerebellum (Fig. 5Ad, 5B).

We also presented birds with playbacks of song while inside our apparatus or the sound isolation chamber. Even in complete darkness, at least half of the birds responded to the song playbacks by hopping excitedly within the confined area, as detected with an infrared camera, and these birds had a similar expression pattern as other hopping birds, including in the areas surrounding the anterior vocal nuclei and lateral to the posterior vocal nuclei (data not shown). In the three birds that remained still, we found robust increased expression in N-L2, including NCM, MV-L2, the HVC shelf, and the RA cup, and other auditory areas as expected [45], but no increased expression in the areas surrounding the anterior vocal nuclei (ASt, AN, and AMV), the areas lateral to the posterior vocal nuclei HVC and RA (DLN and LAI), the somatosensory areas (Nb, MVb, AMD, and AH), or in the cerebellum (Figs. 5Ae, 5B, and 6A). The exception was part of PLN and PLMV; these areas also showed increased robust expression to playbacks of song (Figs. 5Ae, 5B, and 6A).

To further test whether the induced gene expression in these 11 cerebral areas can be independent of auditory input, as is singing-induced gene expression in vocal nuclei [33], we deafened zebra finches and placed them in complete darkness in the rotating wheel. These birds hopped similarly as hearing intact animals. Relative to the hearing intact animals, deafening eliminated the induced expression in all the auditory areas around L2 (N-L2, MV-L2, and NCM), the HVC shelf, and the RA cup (Figs. 5Af, 5B, and 6C) supporting previous findings that these regions are predominantly auditory [23], [33], [45]. But, these deafened hopping birds still showed strong induced gene expression in the areas surrounding the anterior vocal nuclei (ASt, AN, and AMV), in all four areas lateral to the posterior vocal nuclei (DLN, LAI, PLN, and PLMV), the somatosensory areas (Nb, MVb, AMD, and AH), and the cerebellum (Figs. 5Af, 5B, and 6C); this is the most distinct movement-associated expression pattern that we have seen. We conclude that the induced expression in the auditory areas of moving animals is a result of the animals hearing movement- and/or mechanically-generated sounds, and the remaining areas have movement-associated gene expression that is independent of auditory input. We also conclude that the same parts of PLN and PLMV can independently have movement-induced and hearing-induced gene expression.

We noticed that although ZENK expression in vocal nuclei was much lower than in the adjacent brain areas of the moving birds, in some animals there were some cells that expressed ZENK (Fig. 5Ab,c,f). Thus, we quantified ZENK expression in Area X and HVC of all birds and found a trend of low-level increased expression that approached significance in some of the moving groups (Fig. 5B). Future experiments will be needed to determine if this increase is real and associated with limb and body movements or whether some birds produce some vocalizations while hopping.

Physical relationship to vocal nuclei.

To determine how close the movement-associated areas are to the vocal nuclei and to clarify the relative physical relationship of the posterior vocal nuclei to auditory areas, we quantified the distances in Nissl stained serial sagittal (Figs. 6 and 7) and coronal (Fig. S3) sections of the deaf, dark, hopping animals. We used serial sections of hearing song-induced and singing-induced IEG expression in other birds, along with FoxP1 expression in adjacent sections, to help identify the auditory regions, the vocal nuclei, and cerebral subdivision boundaries (Figs. 6 and 7; Fig. S3; see methods). The ZENK activated neurons of ASt, AN, and AMV were on average only ~11, 28, and 10 µm distant from the edge of vocal nuclei Area X, LMAN, and MO respectively (range 0–30 µm, Table 2). That is, there were either no or 1–3 unlabelled neurons between the movement-activated neurons and these vocal nuclei. This expression within ASt, AN, and AMV surrounded the central portions of the anterior vocal nuclei, was most prominent anterior and ventral to them (Fig. 7A, medial), and was less on their caudoventral sides further laterally (Fig. 7A, lateral). This pattern suggests a topographical relationship in the functional activation across brain subdivisions.

Figure 7. High power images of IEG activation in zebra finch vocal nuclei during singing and in adjacent movement-associated areas during hopping.

(A) Anterior vocal nuclei adjacent to ASt, AN, and AMV. (B) HVC adjacent to DLN and dorsal PLN. (C) NIf and Av adjacent to ventral PLN and PLMV respectively. (D) RA adjacent to LAI. The c-fos expression in vocal nuclei (first column) is of a young zebra finch male that sang for 30 min while moving relatively little; c-fos is shown for its high contrast in vocal nuclei relative to the surrounding non-vocal areas. The hopping-associated expression (left two columns) is from a male that hoped in the dark and was deaf; the left most sections are lateral to the vocal nuclei (except for anterior areas, which still contain the lateral part of the anterior vocal nuclei). Yellow dashed lines-brain subdivision boundaries; white dashed lines–vocal nuclei boundaries, only highlighted for some images so that other sections can be viewed as is. Anterior is right, dorsal is up–sagittal sections; sections of the top panel are orientated at a ~45° angle so that all three anterior vocal nuclei fit vertically into one image. Scale bar, 200 µm.

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Table 2. Distances between movement-associated areas and vocal nuclei

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For the posterior areas, the dorsal part of PLN was ~10 µm ventral and anterior to the lateral ~1/3^rd of HVC (Fig. 7B; brightfield in Fig. S4Ab), whereas DLN began ~88 µm posterior to HVC and increased in size further laterally and posteriorly (Fig. 7B; Fig. S4Ac; coronal in Fig. S3Ba). The auditory shelf directly ventral to the medial ~2/3^rds of HVC that shows auditory-induced expression (Fig. 6A, 3^rd and 4^th row) did not show any clusters of labeled neurons in the hopping deaf animals (Fig. 6C; Fig. S4Aa). For the location of PLN and PLMV relative to NIf and Av, it was simple to identify the vocal nuclei in singing animals using IEG expression (Fig. 7C), but difficult to identify by Nissl alone in the hopping animals. However in the hopping animals, we noted a split of expression within PLN where NIf (but also adjacent field L2) would be located and a split between PLN and PLMV by the mesopallium lamina (LM, Fig. 7C, medial; Fig S4Bb). To determine if the first split was due to NIf, we false colored the IEG expression from singing and hopping deaf animals, aligned them by the LM boundary, and found that PLN appeared to surround the dorsal ~1/3^rd of NIf and that PLMV was directly dorsal to Av (Fig. S4D). Further lateral, when NIf was no longer present, the split in PLN was no longer present (Fig. 7C, lateral; Fig. S4Bc). Further medial, field L2 was easily seen by its smaller size neurons relative to L1 and L3, but there was no movement-associated expression around it (Fig. S4Ba). Using these alignments, we calculated that PLN and PLMV are on average ~8 and 17 µm from the NIf and Av respectively (Table 2). For LAI, it was ~28 µm caudal to the lateral ~1/3^rd of RA (Fig. 7D, medial; Fig. S4Cb, Table 2), and wrapped around RA laterally such that it occupied a similar central position in the arcopallium (Fig. 7D, lateral; Fig. S4Cc; coronal in Fig. S3Bb). There was also some expression in the arcopallium caudoventral to RA (Fig. 7D; Fig. S3Bb). The auditory RA cup anteroventral to the medial ~2/3^rds of RA that shows hearing-induced expression (Fig. 6A, 4^th row) did not show detectable clusters of movement-activated neurons (Fig. 6C; Fig. S4Ca). In contrast to these distances, the known somatosensory areas with movement-associated expression (Nb and MVb) were at a minimum ~962 and ~1,167 µm distant to their nearest vocal nuclei MAN and MO, respectively (Table 2). We did not measure distances for the other two known somatosensory areas (AMD and AH), as there are no known vocal nuclei in their respective brain subdivisions (MD and H). Based on these results, it is clear that 7 of the 11 movement-associated cerebral areas are directly adjacent to the 7 known vocal nuclei; the remaining 4 brain areas not adjacent to the vocal nuclei are known somatosensory regions.

Relative cerebral volume.

We next made a gross assessment of the relative cerebral volume activated during hopping. We found that the movement-associated areas in the deaf, dark, hopping animals comprised ~8.7% of the cerebrum volume (Fig. 8A, total). The largest contributor to this volume was the ASt around Area X and the somatosensory AH within the hyperpallium (Fig. 8A). In comparison, the song nuclei combined comprised ~2.5% of the cerebrum volume, with Area X the largest contributor (Fig. 8A). Most of the movement-associated areas were proportionally larger than the adjacent vocal nuclei; LAI and RA were the exceptions, which were about the same size. In this regard, there was a significant positive correlation between vocal nuclei size and adjacent movement-associated region size (Fig. 8B). This distinct hopping-induced expression pattern contrasts with the pattern found in birds taken from our aviary in the early morning, after 60 min of lights, hearing songs and calls, feeding, flying, hopping, and making physical contact with other birds, which results in widespread ZENK expression in the cerebrum (Fig. S5A). Based on these results, it becomes clear that the hopping-induced expression pattern comprises distinct domains of the cerebrum (Fig. 6C), most of which are directly adjacent to the vocal nuclei.

Figure 8. Relative volumes of movement-associated areas and vocal nuclei.

A. Relative volumes as a percentage of the summed cerebral (telencephalon) volume from a series of sagittal sections (see methods) from the hopping, dark and deaf animals. The movement-associated areas and adjacent vocal nuclei are shown as bars adjacent to each other. Totals represent the summed relative volumes of all movement-associated areas whether or not it is adjacent to a vocal nucleus, and of all vocal nuclei. * = p<0.05, paired t-test (within bird comparisons, n = 3). Error bars, S.E.M. Although all animals had higher volumes of ASt and AMV relative to Area X and MO, the variance was large in the movement areas such that the volume difference did not reach significance. B. Correlation between relative volumes of movement-associated regions and adjacent vocal nuclei. Each dot represents the average values from the graph in (A).

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Confirmation with another IEG.

We tested whether movement-associated gene expression was specific to ZENK or a general IEG property, by examining expression of another IEG, c-fos, which is generally thought to be less sensitive to small changes in neural activity, and thus can reveal areas that were highly active during a specific behavior. We found that c-fos was induced in the same cerebral brain areas adjacent to the vocal nuclei as well as in known somatosensory areas (Fig. 9), but with a higher contrast of activation relative to ZENK (Fig. 6C). As with ZENK, the c-fos induction in these dark and deaf animals was independent of visual and auditory input.

Figure 9. Movement-induced c-fos expression in zebra finches.

A. Vocal areas: brain section containing all seven cerebral vocal nuclei from a singing bird. B. Movement areas: serial sections from a hopping bird in the rotating wheel in the dark while deaf. The patterns are similar to that found with ZENK (Fig. 6C). See figure 6E for delineation of anatomical boundaries. Anterior is right, dorsal is up. Scale bar, 2 mm.

doi:10.1371/journal.pone.0001768.g009

Summarizing Part I of this study, in songbirds, 11 cerebral areas showed movement-associated activation that can be grouped into 4 clusters: an AH and AMD cluster comprising known subdivisions of a somatosensory pathway; a Nb and MVb cluster within known subdivisions of a second somatosensory pathway; an anterior ASt, AN, and AMV cluster surrounding the three anterior vocal pathway nuclei Area X, MAN, and MO, respectively; and a posterior DLN, LAI, PLN, and PLMV cluster posterior-laterally adjacent to the four posterior vocal pathway nuclei HVC, RA, NIf, and Av respectively. We do not know if the movement-associated IEG activation adjacent to the vocal nuclei is the result of pre-motor activity as occurs in the vocal nuclei during singing [32]–[34], is the result of somatosensory feedback activity via muscle spindles, or is from a combination of both; thus, we call the induced gene expression as movement-associated. Regardless of the specific source of activation, these findings led us to hypothesize that vocal learning systems may have evolved out of a pre-existing motor or somatosensory-motor system, which may explain the anatomical similarities among distantly related vocal learning birds. If true, then the other vocal learning birds should show a similar relationship between movement-associated areas and their cerebral vocal nuclei, an idea we tested next.

Part II. Movement-Associated Brain Areas in Other Vocal Learners

Parrots.

In parrots, the three anterior vocal pathway nuclei are situated in nearly identical brain locations as their analogous counterparts in songbirds, but the counterparts of the four songbird posterior vocal nuclei are situated much further (1000s of µm) laterally away from the main avian auditory pathway areas (Fig. 1B, yellow). Therefore, this differential relationship provides a natural experiment to test whether movement-associated brain areas would be adjacent to parrot vocal nuclei or instead in the same location as in songbirds, near the auditory pathway. To perform this test, we placed deafened budgerigars (Melopsittacus undulatus, a small parrot) in the rotating wheel in complete darkness. The parrots performed hopping movements similar to zebra finches, and in addition often bobbed the head before a hop. We compared brain ZENK expression in these birds with sitting or hopping animals in dim light, and with hearing intact animals that either heard or produced parrot warble song (Fig. 10Aa,b) [6].

Figure 10. Movement-induced ZENK expression in budgerigars, a parrot.

A. Serial sagittal sections of: (a) Auditory areas: bird sitting relatively still while hearing playbacks of budgerigar warble song; (b) Auditory and vocal areas: perched bird hearing himself and producing warble song while alone and moving relatively little; (c) Movement areas: bird hopping in the rotating wheel in the dark while deaf; (d) FoxP1 expression from adjacent sections of the bird in (c); (e) Corresponding anatomical drawings; red: areas with movement-induced expression; blue: areas with auditory- or visual-induced expression. First row are medial-most sections. Sections with N-L2 are not shown. Anterior is right, dorsal is up. Scale bar, 2 mm. Compare with frontal sections in figure S6. B. Quantification of ZENK expression levels in 22 different brain regions in three groups of budgerigars. * = p<0.05 to<0.0001, one-way ANOVA followed by Holm-Sidak multi-comparison test relative to the sitting still group (n = 3/group). t = significantly different, p<0.05, by a t-test. (t) = 0.06<p<0.09 by a t-test. Although the cerebellum lobule VI clearly had high levels of induced expression in all three animals of the hopping, dark, deaf group, this difference approached significance (p = 0.06) due to a large variance; the same was true of the PH region in the hopping, dim light group. Error bars, S.E.M.

doi:10.1371/journal.pone.0001768.g010

Similar to deafened songbirds hopping in darkness, the induced ZENK and c-fos expression patterns in the deafened parrots hopping in darkness were distinct, with the highest levels distributed across at least nine cerebral brain areas of which seven were directly adjacent (within 100 µm) to the seven parrot vocal nuclei (Fig. 10Ac, 10B; c-fos not shown). Figure S6 shows coronal sections for comparison. These brain areas were: ASt, AN, and AMV with the highest expression directly caudal and medial to the parrot anterior vocal nuclei MMSt, NAO, and MO (analogs of songbird Area X, MAN, and MO respectively); the lateral nidopallium (LN) surrounding LAN (proposed analog of songbird NIf) and the adjacent lateral ventral mesopallium (LMV) surrounding LAM (proposed analog of songbird Av); the LAI surrounding AAC (analog of songbird RA); and the supra-lateral nidopallium (SLN, for details see anatomy section in methods) surrounding NLC (analog of songbird HVC) (Fig. 10Ac, 10B; Fig S6B). For each of these posterior areas (LN, LMV, LAI, and NLC), expression was highest dorsally and/or posteriorly to the vocal nuclei. Also as in songbirds, the somatosensory areas of AMD and AH showed robust induced expression (Fig. 10Ac, 1^st row and 10B; Fig S6B, 1^st row). There was activation in the presumed second somatosensory areas Nb and MVb adjacent to B, but these two areas are also adjacent to two of the posterior vocal nuclei of parrots (LAN and LAM) making it difficult to parse out the boundaries, if any (Fig. 10Ac, 2^nd row and 10B; Fig S6B, 2^nd row). There were no detectable high levels of induced expression in the known auditory areas (N-L2, NCM) or in the posterior dorsal nidopallium (PDN) where songbird DLN and HVC would be expected to be located (Fig. 10A,B) if they were in the same relative location to the auditory pathway as in songbirds. The anterior vocal nuclei, such as MMSt also did not show induction (Fig. 10Ac, 10B). In hearing intact hopping budgerigars in dim light, induced expression occurred in the same brain areas as well as in visual (PMD, PH, Ne, and MVe) and auditory (N-L2) areas (Fig. 10B) similar to that found in songbirds, presumably due to optic flow in dim light and to the animals hearing the hops and/or the mechanical rotating sounds of the wheel, respectively. In both moving groups, there was induced expression in the anterior half of the cerebellum (highest in VI) and in lobule IXcd (Figs. 4G and 10B), similar to that seen in zebra finches that hopped in the wheel (Fig. 4F).

Hummingbirds.

We next investigated the Anna's hummingbird (Calypte anna), a known vocal learner [48]. The three anterior vocal pathway nuclei of hummingbirds are situated in nearly identical brain locations as in songbirds and parrots, but the proposed counterparts of the four posterior vocal nuclei are situated at intermediate caudal-lateral locations, partly adjacent to the auditory pathway areas (Fig. 1B, yellow). Attempting to perform similar movement experiments with hummingbirds would not work, because hummingbirds rarely walk or hop for mobility. Instead, they fly, even to move a few centimeters from one perch location to another. Further, when Anna's hummingbirds were placed in darkness for 2–3h to reduce brain IEG expression to basal levels, they went into torpor, a unique hibernation-like sleeping state that makes them immobile [49]. Thus, for the hummingbirds, we plugged one ear with clay and covered the ear and ipsilateral eye with black vinyl tape to reduce visual motion- and auditory wing humming-induced gene expression in one hemisphere. Covering one eye in songbirds causes ZENK expression to be reduced in visual pathways of the contralateral hemisphere [27] (Hara, Kubikova, Hessler, Jarvis; submitted), as in laterally eyed birds the visual pathways are nearly completely crossed [50]. Auditory input is bilateral, but removing input from one ear in chicks causes some auditory nuclei to have reduced 2-deoxyglucose activation in the contralateral hemisphere [51], [52]. We then placed the hummingbirds in dim light inside a plexiglass box with a central perch (see methods). Most Anna's hummingbirds remained relatively still on the perch for the first several hours; thereafter, some birds performed stereotypical circular hovering flights on and off the perch in the direction of the open eye while others remained perched, relatively still (less than ~15 flights in 60 min), and awake.

In the hovering hummingbirds, there was an induced ZENK expression pattern that was discrete and significantly different between hemispheres, which allowed us to suggest which brain areas have movement-associated versus visual- and possible auditory-associated gene expression (Fig. 11Ab). Figure S7 shows serial coronal sections for comparison. Apparent visual pathway areas (PMD, PH, Ne, MVe, and optic tectum [OT]) showed increased expression in the animals that made hovering flights, but this expression was significantly reduced in the hemisphere contralateral to the covered eye (Fig. 11Ab, 11B; Fig. S7B). A similar and visible, but non-significant trend was seen in the auditory region N-L2 (including NCM; Fig. 11Ab, 11B; Fig. S7B), perhaps due to crossed pathways. In contrast, there was equivalent bilateral increased expression in hovering birds within the ASt, AN, and AMV medial to and surrounding the anterior vocal pathway nuclei VASt, VAN, and VAM (analogs of songbird Area X, MAN, and MO, respectively), in the DLN medially adjacent to the VLN vocal nucleus (analog of songbird HVC), and in the LAI caudally and ventrally adjacent to the VA vocal nucleus (analog of songbird RA; Figs. 11Ab, 11B; Fig. S7B; higher power images shown in Fig. 12A–C). Although hummingbird DLN expression was distinctly medially adjacent to VLN instead of lateral to it as in songbirds, the hummingbird VLN is positioned more laterally than the songbird analog HVC and thus the DLN area of movement-associated activation is in a similar location as that of songbirds (Fig. 12B vs Fig. S3Ba). As in songbirds and parrots, these areas of apparent movement-associated gene expression were less than 100 µm distant to the vocal nuclei.

Figure 11. Movement-induced ZENK expression in male Anna's hummingbird.

A. Serial sagittal sections of: (a) Vocal and other areas: ZENK expression in a bird that was singing interspersed with flying near an outdoor feeder in the morning (high expression in non-vocal areas is due to other behaviors, including flying and feeding); (b) Auditory, visual, and movement-associated areas: ZENK expression in control hemisphere (contralateral to open eye and ear) and experimental hemisphere (contralateral to covered eye and ear) of a bird hovering in a plexiglass cage in dim light; (c) FoxP1 expression from adjacent sections of the experimental hemisphere of the bird in (b); (d) Corresponding anatomical drawings; red: areas with movement-induced expression; blue: areas with visual- or auditory-induced expression (auditory areas also determined from a previous study [7]). First row are medial-most sections. Anterior is right, dorsal is up. Scale bar, 2 mm. Compare with coronal sections in figure S7. B. Quantification of ZENK expression levels in 18 different brain regions, in both hemispheres, in two groups of hummingbirds. * indicates brain areas with statistically significant increases in both experimental (covered) and control (open) hemispheres of hovering birds relative to the experimental and control hemispheres of the relatively still birds (p<0.05, one-tailed t-test, n = 3 relatively still and 4 hovering animals for both hemispheres). # indicates significantly less increase in the experimental hemisphere (p<0.05, paired t-test on experimental and control hemispheres within birds). The (*) for the OT indicates that this is the only visual area that did not show increased ZENK expression in the experimental hemisphere opposite the covered eye. Error bars, S.E.M.

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Figure 12. High power images of IEG activation in hummingbird vocal nuclei during singing and in adjacent movement-associated areas during hopping.

A. Anterior vocal nuclei adjacent to ASt, AN, and AMV in sagittal sections. B. DLN adjacent to VLN [and LAI to VA] in coronal sections. C. LAI adjacent to VA [and DLN to VLN] in sagittal sections. D. VMN [as well as VMM] adjacent to activated areas near L2 in sagittal sections. The c-fos expression in vocal nuclei (first column) is of male that sang for 30 min interspersed with flying and feeding; c-fos is shown for its high contrast in vocal nuclei relative to the surrounding non-vocal areas. The hovering-associated expression patterns (left two columns) are from the hemisphere opposite of the covered eye and ear of males that hovered in a plexiglass cage. Anterior is right, dorsal is up for sagittal sections; medial is left, dorsal is up for frontal sections. The left most sections are either lateral (A, C, and D) or caudal (B) to that shown in the middle column. Different background red color is due to different cresyl violet staining intensities. Note that similar to budgerigar MO and NAO (Fig. 10Ab and Fig. S6A), the two analogous hummingbird pallial anterior vocal nuclei (VAM and VAM) are very close such that the IEG expression does not distinguish the brain subdivision boundary well. Yellow dashed lines-brain subdivision boundaries; white dashed lines–vocal nuclei boundaries, only highlighted for some images so that other sections can be viewed as is; boundaries were determined from Nissl stain and adjacent sections hybridized with FoxP1 (Fig. 11Ac and Fig. S7C). Scale bars, 200 µm.

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There was also increased bilateral expression in the apparent somatosensory areas AMD and adjacent AH (Fig. 11Ab top three rows, 11B and 12A). We did not note high expression levels in the second somatosensory areas Nb and MVb. We could not reliably determine whether there was induced expression adjacent to the other two posterior vocal nuclei VMN and VMM (proposed analogs of songbird NIf and Av) in the medial nidopallium and mesopallium respectively, because these nuclei in hummingbirds, more so than in songbirds, are relatively small and difficult to locate without singing-induced gene expression; likewise, they are also directly adjacent to auditory areas. Nevertheless, in the several birds where we were able to locate these nuclei, there was still adjacent high induced ZENK expression bilaterally, but in regions that, like songbird PLN and PLMV, also show hearing-induced gene expression (Fig. 11A top row, and 12D [7]). The cerebellum had increased activation in lobules V and IXcd (Fig. 4H), similar, although not identical, to the pattern seen in garden warblers that performed wing whirring (Fig. 4C); hummingbirds have under-developed lobules II and III, which are thought to modulate leg movements [53] (but see discussion). Lobule VI already had high expression in the relatively still animals that performed few flights (Fig. 11B).

Summarizing part II of this study, all seven parrot and at least most hummingbird cerebral vocal nuclei are directly adjacent to areas that are activated during movement, even though some of the posterior vocal pathway nuclei are in different brain locations in each group. This activation does not require visual or auditory input. These findings suggest that vocal learning systems in distantly related birds are adjacent to a pre-existing system involved in movement control. If true, then vocal non-learning birds should have similar brain areas associated with movement control, an idea that we tested next.

Part III. Movement-Associated Brain Areas in Vocal Non-Learners

Female songbirds.

Females of many songbird species, including zebra finches, do not have vocal learning behavior, i.e. song or learned calls [12]. Their forebrain vocal nuclei (except for LMAN) are atrophied as seen in zebra finch females [54] and garden warblers (noted here). We separated out the females in the garden warbler groups described above (n = 10) and found that in those that performed movement behaviors (wing whirring and flights) ZENK gene activation was present within comparable cerebral areas and in the cerebellum as seen in males, but without the presence of negative expression regions of vocal nuclei (Fig. S8A, except for LMAN). To determine a more restricted pattern, we analyzed ZENK expression in the brains of deafened female zebra finches placed in the rotating wheel in the dark. Movement-induced ZENK expression was found in the same 11 cerebral brain areas and in the cerebellum as in males, except that the expression where the anterior vocal nuclei would be expected to be located was patchier and diffuse (Fig. S8B ;PLN, PLMV, and LAI not shown; p<0.02 hopping females [n = 4] relative to sitting males [n = 6]; p>0.05 hopping females [n = 4] relative to hopping males [n = 3] for all 11 areas, two-tailed t-test). Thus, these movement-associated areas appear to be present independent of the presence of functioning vocal nuclei. However, female songbirds of an ancestral species may have once had vocal learning behavior and associated brain nuclei that were then subsequently lost in some species. Thus, to test our hypothesis further, we examined movement-associated gene expression in a vocal non-learning species.

Ring Doves.

Ring doves (Streptopelia risoria) are interrelated between songbirds and hummingbirds (Fig. 1A). Nevertheless, they are known vocal non-learners [55] and do not possess cerebral vocal nuclei [15]. As ring doves are bigger than zebra finches and budgerigars, they did not fit into our rotating wheel apparatus. Further, ring doves do not normally hop when they move from one nearby location to another, but instead walk. Therefore, we placed deafened ring doves in darkness on a treadmill designed for animals the size of rats and compared their gene expression to intact controls that sat still (Fig. 13A,B).

Figure 13. Movement-induced ZENK expression in ring doves, a vocal non-learner.

A. Sagittal sections of: (a) Dove sitting relatively still in the dark; (b) Dove walking on a treadmill in the dark while deaf; (c) FoxP1 expression from adjacent sections of the bird in (b); (d) Corresponding anatomical drawings; red: areas with movement-induced expression; blue: known auditory and visual areas. First row are medial-most sections. Front is right, dorsal is up. Scale bar, 2 mm. B. Quantification of ZENK expression levels in 20 different brain regions in two groups of ring doves. * = p<0.05 to<0.0001, one-tailed t-test, relative to sitting still animals (n = 3/group). Error bars, S.E.M.

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We found that in sitting controls, there was low ZENK expression throughout the brain except in the auditory pathway, which had unusually high basal levels that were partly reduced by deafening (Fig. 13Aa,b). The olfactory bulb also had high basal levels. When the treadmill floor moved, the doves walked. ZENK expression in these doves was induced to high levels in anterior cerebellum lobules I-VI and posterior lobule IXcd (Fig. 4I), similar, although not identical to that seen in songbirds and budgerigars that hopped (Fig. 4F, G). In addition, there was increased expression in somatosensory areas of AMD and adjacent AH, and Nb and adjacent MVb (Fig. 13Ab, 13B; but more widespread in Nb and MVb, not shown) similar to that seen in songbirds, parrots, or hummingbirds (except for Nb and MVb in the latter). When we examined expression in the anterior medial forebrain where we would expect to find anterior vocal nuclei in vocal learners, there was increased expression in ASt, AN, and AMV without the presence of negative expression regions of vocal nuclei (Fig. 13Ab, 13B). The expression was more diffuse as seen in female zebra finches. In the posterior cerebrum, there was increased expression in the DLN region lateral to where we would expect to find songbird HVC, and in the LAI region lateral to where we would expect to find songbird RA (Fig. 13Ab, 13B); in ring doves, the location of the arcopallium relative to the rest of the cerebrum is intermediate in medial-lateral position between that of songbirds and parrots. Finally, there was higher expression in the PLN and adjacent PLMV dorsolateral to where we would expect to find songbird NIf and Av, but the relative increases were not as large due to the unusually high basal levels of ZENK expression in these and the adjacent auditory pathway areas of sitting hearing intact animals (Fig. 13Ab, 13B). Nevertheless, the increase was independent of auditory and visual input, since the birds were deafened and in darkness. In the olfactory bulb, there was no significant difference between sitting still and walking animals (p>0.05, one-tailed t-test).

Summarizing part III of this study, vocal non-learners appear to have movement-associated gene expression in brain areas similar to that of vocal learners. The expression patterns in the cerebellum and AH and AMD somatosensory areas are quite similar across the vocal learning and vocal non-learning groups. The expression patterns in vocal non-learners where one would expect to find vocal nuclei are more uniform without the presence of negative regions due to vocal nuclei.

Discussion

This is the first study we are aware of to globally map movement-associated areas in the avian cerebrum. The discovered areas are adjacent to the cerebral vocal nuclei in all three vocal learning orders. The anatomical extent of the movement-associated areas are larger than the vocal nuclei, which is consistent with a greater amount of musculature involved in the control of limb and body movements relative to that for the syrinx. Below we discuss the implications of our results for understanding motor and somatosensory pathways in birds, and the evolution of brain pathways for vocal learning.

Movement-associated brain areas in birds

It is well established that voluntary movements in mammals are controlled by motor and somatosensory pathways in the cerebrum. Motor cortical areas send commands to lower motor neurons in the brainstem and spinal cord that control muscle contraction and relaxation, and to motor basal ganglia areas that modulate ongoing movements, whereas muscle spindles send proprioceptive feedback to somatosensory areas that sense and modulate ongoing movements [56]. However, the motor and somatosensory pathways function in an overlapping manner: the somatosensory cortex sends efference copies of sensory commands to motor cortex prior to the motor command, and the motor cortex has inhibitory connections with the somatosensory cortex to modulate the somatosensory input [57]. Although cerebral motor pathways are well understood in the mammalian brain, surprisingly little is known for the avian brain. Here we found that limb and body movements result in activation within specific cerebral areas of the two known somatosensory pathways (AH and AMD; Nb and MVb). There was striking specificity in the activation patterns when other sensory factors (vision and audition) were eliminated, allowing us to map functional domains of the avian cerebrum. Although such specificity cannot be readily revealed by tracer studies, such studies have shown that the AH area of zebra finches, owls, and pigeons receives input from AMD and from the anterior portion of the intercalated lamina of the hyperpallium (IH), which in turn receives input from the somatosensory thalamus and spinal cord dorsal column nuclei, which are innervated by somatosensory neurons from the wings and legs [58]–[61]. AH also sends descending projections to the intermediate grey matter of the spinal cord [59]. Some have interpreted this pyramidal tract-like projection to the spinal cord to indicate that AH may also have motor or mixed somatosensory-motor functions, and is the homolog of the mammalian motor cortex [62]. Wild and Williams [59] who discovered this projection proposed instead that it is not motor, but somatosensory feedback to the ascending somatosensory pathway of the spinal cord. As for the other pathway, B also receives somatosensory input and projects to Nb [63] and Nb projects to MVb [64]; it is not clear where MVb projects to. Electrophysiology studies show that B has a somatotopic map of the body in budgerigars [65], whereas AH has a touch somatotopic map of the leg and foot in owls [60]. Presumably the areas that sense leg movements during hopping or wing movements during whirring were specifically activated in our study. This activation could conceivably be used for processing proprioceptive feedback from muscles spindles and/or skin touch receptors as the animal sense the floor with its feet or surrounding air with its wings, an idea that can be tested with peripheral stimulation and removal of somatosensory input.

There was also consistent cerebellum activation, as would be expected because the cerebellum receives somatosensory input and sends motor output commands for fine coordination of movements [53], [66], [67]. Our findings are the first that we are aware to identify patterns of movement-associated IEG activation in the cerebellum. The cerebellum in birds, as in mammals, has two somatotopic body representations: one in the anterior half from lobules I-VI and the other in posterior lobules IX-X [67]; determining more specific topographic organization has yielded conflicting results [53]. Connectivity data in pigeons and zebra finches suggest overlapping zones where lobules I-III and IXab receive input from the neck, III-V from the wings, III-VI and IXcd from the legs, VII-VIII from visual and auditory areas but also from somatosensory AH [66], [67]. Our findings are partly consistent with this picture, in that wing whirring activated IEG expression mostly in lobules II-VI, hopping in lobules VI and IXcd, and when moving in dim light or in the dark little if any activation occurred in VII-VIII. In general, the cerebellum activation patterns suggest that limb movements may be mostly responsible for the overall brain gene activation seen in the controlled hopping movement groups, as lobules II-VI and IXcd that are connected to the limbs were consistently activated.

With known somatosensory areas identified, a remaining question is where are the motor areas? Besides the hypothesis that AH and AMD are motor in addition to somatosensory [62], previous studies have suggested the arcopallium and dorsal striatum as general motor areas of the avian cerebrum [68], [69]. However, none that we are aware of have used movement behavior to map a cerebral motor system. Based on our results, we hypothesize that a general motor system in birds consists of the brain areas adjacent to the cerebral vocal nuclei of vocal learners. Our reasons are as follows: First, we found a close association in locations and size of these movement-associated areas with the cerebral vocal nuclei. Second, like the vocal nuclei [32]–[34], these brain areas have movement-associated IEG expression that is independent of auditory and visual input. Third, like the vocal nuclei, the expression levels correlate with the amount of movement performed. Fourth it appears that the vocal nuclei and adjacent regions have similar connectivity, as described below.

Many prior studies have accidentally or purposely placed tracers adjacent to the songbird vocal nuclei, and except for the HVC shelf and RA cup [23] the function of these brain regions were not known. In some of these studies on zebra finches, we note remarkable overlap in the connectivity patterns [70], [71] with the movement-associated gene expression patterns (this study). When we compile these connectivity results with the movement-associated gene expression results (Table 3 [63], [70]–[74]), it appears that the movement-associated areas in songbirds may be connected in anterior and posterior pathways in parallel, although not identical, with the adjacent vocal nuclei (Fig. 14A). The anterior movement-associated areas, like the anterior vocal pathway nuclei, appear to be connected in a pallial-basal-ganglia-thalamic-pallial loop (Fig. 14A; white arrows): anterior AMV to AN adjacent to LMAN, these two areas to the striatum adjacent to Area X, the striatum via its pallidal-like neurons to the dorsal thalamus adjacent to the vocal part of DLM, and the dorsal thalamus back to the AN adjacent to LMAN. Connectivity of MO or the surrounding MV is not known in songbirds, but the comparable song nucleus and adjacent MV in parrots and the MV in pigeons projects to the anterior nidopallial and striatal vocal nuclei and surrounding area, respectively [75], [76]. The parrot anterior vocal pathway also forms a pallial-basal-ganglia-thalamic-pallial loop (Fig. 1B) [76]. That is, similar connectivity can be compiled for these cerebral regions in other vocal learning and vocal non-learning birds [25].