Significance Birds are remarkably intelligent, although their brains are small. Corvids and some parrots are capable of cognitive feats comparable to those of great apes. How do birds achieve impressive cognitive prowess with walnut-sized brains? We investigated the cellular composition of the brains of 28 avian species, uncovering a straightforward solution to the puzzle: brains of songbirds and parrots contain very large numbers of neurons, at neuronal densities considerably exceeding those found in mammals. Because these “extra” neurons are predominantly located in the forebrain, large parrots and corvids have the same or greater forebrain neuron counts as monkeys with much larger brains. Avian brains thus have the potential to provide much higher “cognitive power” per unit mass than do mammalian brains.

Abstract Some birds achieve primate-like levels of cognition, even though their brains tend to be much smaller in absolute size. This poses a fundamental problem in comparative and computational neuroscience, because small brains are expected to have a lower information-processing capacity. Using the isotropic fractionator to determine numbers of neurons in specific brain regions, here we show that the brains of parrots and songbirds contain on average twice as many neurons as primate brains of the same mass, indicating that avian brains have higher neuron packing densities than mammalian brains. Additionally, corvids and parrots have much higher proportions of brain neurons located in the pallial telencephalon compared with primates or other mammals and birds. Thus, large-brained parrots and corvids have forebrain neuron counts equal to or greater than primates with much larger brains. We suggest that the large numbers of neurons concentrated in high densities in the telencephalon substantially contribute to the neural basis of avian intelligence.

Many birds have cognitive abilities that match or surpass those of mammals (1). Corvids and parrots appear to be cognitively superior to other birds, rivalling great apes in many psychological domains (1⇓–3). They manufacture and use tools (4, 5), solve problems insightfully (6), make inferences about causal mechanisms (7), recognize themselves in a mirror (8), plan for future needs (9), and use their own experience to anticipate future behavior of conspecifics (10) or even humans (11), to mention just a few striking abilities. In addition, parrots and songbirds (including corvids) share with humans and a few other animal groups a rare capacity for vocal learning (12), and parrots can learn words and use them to communicate with humans (13).

Superficially, the architecture of the avian brain appears very different from that of mammals, but recent work demonstrates that, despite a lack of layered neocortex, large areas of the avian forebrain are homologous to mammalian cortex (14⇓–16), conform to the same organizational principles (15, 17, 18), and play similar roles in higher cognitive functions (14, 19), including executive control (20, 21). However, bird brains are small and the computational mechanisms enabling corvids and parrots to achieve ape-like intelligence with much smaller brains remain unclear. The notion that higher encephalization (relative brain size deviation from brain–body allometry) endows species with improved cognitive abilities has recently been challenged by data suggesting that intelligence instead depends on the absolute number of cerebral neurons and their connections (22⇓⇓–25). This is in line with recent findings that absolute rather than relative brain size is the best predictor of cognitive capacity (26⇓–28). However, although corvids and parrots feature encephalization comparable to that of monkeys and apes, their absolute brain size remains small (29, 30). The largest average brain size in corvids and parrots does not exceed 15.4 g found in the common raven (29) and 24.7 g found in the hyacinth macaw (30), respectively. Do corvids and parrots provide a strong case for reviving encephalization as a valid measure of brain functional capacity? Not necessarily: it has recently been discovered that the relationship between brain mass and number of brain neurons differs starkly between mammalian clades (31). Avian brains seem to consist of small, tightly packed neurons, and it is thus possible that they can accommodate numbers of neurons that are comparable to those found in the much larger primate brains. However, to date, no quantitative data have been available to test this hypothesis.

Here, we analyze how numbers of neurons compare across birds and mammals (32⇓⇓⇓⇓⇓⇓–39) of equivalent brain mass, and determine the cellular scaling rules for brains of songbirds and parrots. Using the isotropic fractionator (40), we estimated the total numbers of neuronal and nonneuronal cells in the cerebral hemispheres, cerebellum, diencephalon, tectum, and brainstem in a sample of 11 parrot species, 13 vocal learning songbird species (including 6 corvids), and 4 additional model species representing other avian clades (Figs. S1 and S2). Because most of the cited mammalian studies analyzed cellular composition of only three brain subdivisions, namely the pallium (referred to as the cerebral cortex in those papers), the cerebellum, and rest of brain, we divided the avian brain identically to ensure an accurate comparison of neuronal numbers, densities, and relative distribution of neurons in birds and mammals. Specifically, the avian pallium (comprising the hyperpallium, mesopallium, nidopallium, arcopallium, and hippocampus) was compared with its homolog—the mammalian pallium (comprising the neocortex, hippocampus, olfactory cortices such as piriform and entorhinal cortex, and pallial amygdala) (14⇓–16, 41). The avian subpallium (formed by the striatum, pallidum, and septum), diencephalon, tectum, and brainstem were pooled and compared with the same regions of mammalian brains that are referred to as “the rest of brain.” The cerebellum is directly compared between the two clades. The results of our study reveal that avian brains contain many more pallial neurons than equivalently sized mammalian brains.

Fig. S1. Phylogenetic relationships among the 28 species examined. The tree was constructed using birdtree.org/; its topology follows recent studies (46⇓⇓–49). Note that songbirds and parrots are sister groups and together with the distantly related barn owl belong to the clade core landbirds (Telluraves); the pigeon represents the Columbea, a basal clade of the Neoaves; the red junglefowl represents the Galloanseres, a sister group of Neoaves and the most basal clade of Neognathae; and the emu represents Paleognathae (tinamous and flightless ostriches), the most basal clade of extant birds (48). Also note that all passerine birds examined were vocal learners belonging to the clade Oscines.

Fig. S2. Brain dissection and labeling of neurons and nonneuronal cells. (A and B) Brain of the raven before and after the dissection. (A) Ventral side of the brain showing approximate lines of dissection of the brainstem and tectum. (B) Brain dissected into parts used for isotropic fractionation. (C) NeuN-immunolabeled transverse section of the zebra finch brain depicting the line of dissection of the tectum from the rest of the mesencephalon. (D–F) Dissection of the telencephalon into pallium and subpallium. NeuN-immunolabeled transverse sections of the zebra finch brain at rostral (D), intermediate (E), and caudal (F) telencephalic levels. Lines of dissection follow the pallial-subpallial lamina and divide the telencephalon into pallium (dorsal part) and subpallium (ventral part). Coordinates anterior to the Y point are indicated in millimeters at Bottom Left (64). (G–I) High-power micrographs showing a sample of homogenate from the telencephalon of the Eurasian jay; dissociated nuclei stained with DAPI (G) and immunolabeled with NeuN antibody (H), dual-fluorescence merge image (I). Note that neurons are double-labeled, whereas the nonneuronal cells are devoid of anti-NeuN immunoreactivity. [Scale bars: 10 mm (A and B); 1 mm (C and F); 50 µm (I).]

Discussion Assuming that brains of parrots and songbirds have diverged from the presumptive ancestral avian pattern found in all representatives of basal bird lineages examined and characterized by a mammal-like numerical preponderance of cerebellar neurons, we suggest that birds generally have higher neuronal densities than mammals, and further that parrots and songbirds have acquired an expanded telencephalon with increased neuronal densities. Two proximate, synergistic mechanisms likely contributed to this evolutionary process. First, just like the expansion of neocortex in primates (52), the expansion of the telencephalon in parrots and songbirds is associated with delayed and protracted neurogenesis, an expanded subventricular zone, and delayed neuronal maturation (53⇓–55). It has been suggested that extensive posthatching neurogenesis and brain maturation promote learning from conspecifics and may have facilitated the emergence of specialized circuits that mediate vocal learning and possibly also other flexible and innovative behaviors (56). Second, analyses of brain gene expression profiles strongly suggest that songbirds and parrots independently evolved vocal learning pathways by duplication of preexisting, surrounding motor circuits (57, 58). Intriguingly, parrot pallial song nuclei underwent a further duplication event to evolve a unique additional circuit, the so-called shell song system, which seems to be particularly well developed in large-brained parrots (45). What ultimate mechanisms drive the evolution of the enlarged, neuron-rich telencephalon, which sets parrots and songbirds apart from the more basal birds we examined, remains poorly understood. We suggest that this expansion has been due to simultaneous selective pressures on cognitive enhancement and an evolutionary constraint on brain size, which may stem from the constraints on body size imposed by active flight. Altriciality and the extended parental care that has developed in avian ancestors simultaneously relaxed constraints on the duration of ontogenesis, a precondition for telencephalic expansion by the mechanisms described above (56). Moreover, a short neck relative to many other bird lineages may have reduced biophysical constraints on head size (cf. ref. 59). Our finding of greater than primate-like numbers of neurons in the pallium of parrots and songbirds suggests that the large absolute numbers of telencephalic neurons in these two clades provide a means of increasing computational capacity, supporting their advanced behavioral and cognitive complexity, despite their physically smaller brains. Moreover, a short interneuronal distance, the corollary of the extremely high packing densities of their telencephalic neurons, likely results in a high speed of information processing, which may further enhance cognitive abilities of these birds. Thus, the nuclear architecture of the avian brain appears to exhibit more efficient packing of neurons and their interconnections than the layered architecture of the mammalian neocortex. Further comparative studies on additional species are required to determine whether the high neuronal densities and preferential allocation of neurons to the telencephalon represent unique features of songbirds, parrots, and perhaps some other clades like owls, or have evolved multiple times independently in large-brained birds. More detailed quantitative studies should assess the distribution of neurons among various telencephalic regions involved in specific circuits subserving specific functions. The results, combined with behavioral studies, will enable us to determine the causal relationships between neuronal numbers and densities and perceptual, cognitive, and executive/motor abilities, and greatly advance our understanding of potential mechanisms linking neuronal density with information-processing capacity.

Methods Experimental procedures were all approved by the Institutional Animal Care and Use Committee at Charles University in Prague. Altogether, 73 birds belonging to 28 species were used in this study (Table S1). Animals were killed by an overdose of halothane and perfused with 4% (wt/wt) paraformaldehyde. Brains were removed, postfixed for an additional 7–21 d, and dissected into the cerebral hemispheres, cerebellum, diencephalon, tectum, and brainstem. In one individual per species, one hemisphere was dissected into the pallium and the subpallium. In these brain components, the total numbers of cells, neurons, and nonneuronal cells were estimated following the procedure of isotropic fractionation described earlier (40). The reduced major axis regressions to power functions were calculated to describe how structure mass, numbers of cells, and densities are interrelated across species. Analysis of covariance was used to compare scaling among groups (taxonomic orders or brain regions). To compare relative brain size between corvid and noncorvid songbirds, we computed t test on the residuals of a log–log regression of brain mass against body mass (residual brain mass, hereafter). For the comparison with cellular scaling rules reported previously for mammals, the reduced major axis regressions were calculated from quantitative data published for primates (33, 37, 38), rodents excluding the naked mole-rat (32, 39), and artiodactyls (36). In addition, the published quantitative data for Eulipothyphla (34) and Afrotheria (35) were used for comparison in Fig. S5. Further details are provided in Supporting Information.

SI Methods Animals. Three individuals per species were collected with some exceptions for large parrots, two songbird species, and the emu, in which only one or two birds were examined. The following species were purchased from local breeders: all species of parrots, zebra finch, azure-winged magpie, common hill myna, raven, emu, red junglefowl, and barn owl. According to some authors, the genetic integrity of the red junglefowl Gallus gallus is endangered due to hybridization with domestic or feral chickens at the edge of fragmented forests (60). Although we thus cannot exclude admixture of genes from domestic or feral chicken, the red junglefowl used in this study appeared to have a pure wild phenotype. The remaining birds were wild-caught in Czech Republic (Permission No. 00212/CS/2013 and 446/2013). All birds were sexually mature or at least had adult-like size and plumage coloration. We determined the sex of all animals upon dissection and found that we had included both males and females in the analysis. The sample sizes were too small to analyze sex differences. Animals were killed by an overdose of halothane. They were weighed and immediately perfused transcardially with warmed PBS containing 0.1% heparin followed by cold phosphate-buffered 4% (wt/wt) paraformaldehyde solution. Skulls were partially opened and postfixed for 30–60 min, after which brains were dissected and weighed. Brains were postfixed for additional 7–21 d and then dissected. All procedures were approved by Institutional Animal Care and Use Committee at Charles University in Prague, Ministry of Culture (Permission No. 47987/2013) and Ministry of the Environment of the Czech Republic (Permission No. 53404/ENV/13-2299/630/13). Dissection. Brains were dissected into distinct components using the Olympus SZX 16 stereomicroscope. The cerebral hemispheres were detached from the diencephalon by a straight cut separating the subpallium from the thalamus. The tectum (optic lobe) was bilaterally excised from the surface of the brainstem. The excised parts included most of the tectal gray, optic tectum, and torus semicircularis. Both left and right tectum were processed together. The cerebellum was cut off at the surface of the brainstem. Finally, the remaining structures were dissected into diencephalon (rostral part) and brainstem (caudal part) along the plane connecting the posterior commissure dorsally and hypothalamus–mesencephalon boundary ventrally. For most individuals, only one cerebral hemisphere was processed, because in our preliminary studies we detected negligible differences between left and right hemisphere mass and cell numbers. In one individual per species, the second hemisphere was dissected into the pallium and the subpallium. These hemispheres were embedded in agarose and sectioned on a vibratome at 300–500 μm (depending on size of a hemisphere) in the coronal plane. Under oblique transmitted light at the stereomicroscope and with the use of a microsurgical knife (Stab Knife Straight; 5.5 mm; REF 7516; Surgical Specialties Corporation), we manually dissected the pallium from subpallium on each section by cutting along the pallial-subpallial lamina, as defined by Reiner et al. (41). The subpallium included all major subpallial cell groups enumerated therein; the remaining parts of the telencephalon constituted the pallium. The dissected structures were dried with paper towel, weighed, incubated in 30% (wt/wt) sucrose solution until they sank, then transferred into antifreeze (30% glycerol, 30% ethylene glycol, 40% phosphate buffer), and frozen for further processing. Isotropic Fractionator. We estimated total numbers of cells, neurons, and nonneuronal cells following the procedure of isotropic fractionation described earlier (40). Briefly, each dissected brain division was homogenized in 40 mM sodium citrate with 1% Triton X-100 using Tenbroeck tissue grinders (Wheaton). When turned into an isotropic suspension of isolated cell nuclei, homogenates were stained with the florescent DNA marker DAPI, adjusted to a defined volume, and kept homogenous by agitation. The total number of nuclei in suspension, and therefore the total number of cells in original tissue, was estimated by determining density of nuclei in small fractions drawn from a homogenate. At least four 10-µL aliquots were sampled and counted using a Neubauer improved counting chamber (BDH; Dagenham) with an Olympus BX51 microscope equipped with epifluorescence and appropriate filter settings (Olympus filters U-MWU2 for DAPI and U-MWG2 for Alexa Fluor 546-conjugated secondary antibodies); additional aliquots (typically two to five) were assessed when needed to reach the coefficient of variation among counts ≤ 0.15. Once the total cell number was known, the proportion of neurons was determined by immunocytochemical detection of neuronal nuclear marker NeuN (61). This neuron-specific protein was detected by the mouse monoclonal antibody anti-NeuN (clone A60; Chemicon; dilution, 1:800), which was recently characterized by Western blotting with chick brain samples and shown to react with a protein of the same molecular weight as in mammals (62), indicating that it does not cross-react with other proteins in birds. The binding sites of the primary antibody were revealed by Alexa Fluor 546-conjugated goat anti-mouse IgG (Life Technologies; dilution, 1:500). An electronic hematologic counter (Alchem Grupa) was used to count simultaneously DAPI-labeled and NeuN-immunopositive nuclei in the Neubauer chamber. A minimum of 500 nuclei was counted to estimate percentage of double-labeled neuronal nuclei. Numbers of nonneuronal cells were derived by subtraction. Data Analysis. All analyses were performed using average values for each species; variables were log-transformed before the subsequent statistical analyses. Correlations between variables were assessed using nonparametric Spearman rank test. If a significance criterion of P < 0.05 was reached, the reduced major axis regressions were calculated to describe how structure mass, numbers of cells, and densities are interrelated across species. Analysis of covariance (ANCOVA) was used to compare scaling among groups (taxonomic orders or brain regions). The significant interaction between categorical and continuous predictors in the full-factorial ANCOVA demonstrates statistically different slopes of the regression lines among groups and precludes the direct comparison of the magnitude of differences among groups based just on the differences in intercepts. In these cases, the group responsible for the significant interaction was excluded from the ANCOVA model, and, subsequently, the effect of categorical predictor was tested across groups with statistically homogenous slopes, and their differences were compared based on differences in the intercepts. The planned comparisons of least-squares means was used to examine significant pairwise differences. To compare relative brain size between corvid and noncorvid songbirds, we computed t test on the residuals of a log–log regression of brain mass against body mass (residual brain mass, hereafter). For the comparison with cellular scaling rules reported previously for mammals, the reduced major axis regressions were calculated from quantitative data published for primates (33, 37, 38), rodents excluding the naked mole-rat (32, 39), and artiodactyls (36). In addition, the published quantitative data for Eulipothyphla (34) and Afrotheria (35) were used for comparison in Fig. S4. The regressions were calculated using RMA for JAVA 1.21 (63); ANCOVA and t test, using Statistica 10.0 (Stat Soft); and all other analyses were performed in JMP 10.0 (SAS Institute).

SI Results The results of the ANCOVA are summarized below for selected, important comparisons among taxonomic orders and brain regions. They are listed in order, in which they appear in the figures. Ad Fig. 1. (B) Allometric lines for songbirds (green line) and parrots (red line) do not differ from each other [full-factorial ANCOVA, slopes: F (1,20) = 0.537, P = 0.47; intercepts: F (1,20) = 0.580, P = 0.46], but they do differ from allometric lines for mammals [slopes: F (4,40) = 4.290, P = 0.006; intercepts: F (4,40) = 3.595, P = 0.014; post hoc analyses indicate that the regression line for rodents has a different slope and that parrots and songbirds have significantly smaller brains for a given number of neurons than primates and artiodactyls, P < 10−6 for all planned comparisons]. (E) Allometric lines for the taxa examined are significantly different [slopes: F (5,38) = 3.653, P = 0.009; intercepts: F (5,38) = 2.558, P = 0.043; post hoc analyses indicate that the regression line for primates has a different slope and that parrots and songbirds have a significantly higher number of neurons for a given body mass than rodents and artiodactyls, P < 0.001 for all planned comparisons]. Ad Fig. 2. (B and C) Neuronal density varies significantly among principal brain divisions in both parrots [slopes: F (4,45) = 16.2, P < 10−6; intercepts: F (4,45) = 233.0, P < 10−6] and songbirds [slopes: F (4,55) = 14.4, P < 10−6; intercepts: F (4,55) = 523.9, P < 10−6]. (D and E) Comparison of the telencephalon with data pooled for the all other structures examined indicate that nonneuronal cell density is significantly lower in the telencephalon than in the remaining brain divisions in both parrots [slopes: F (1,51) = 0.00, P = 0.995; intercepts: F (1,51) = 58.94, P < 10−6] and songbirds [slopes: F (1,61) = 0.0, P = 0.838; intercepts: F (1,61) = 238.0, P < 10−6]. Ad Fig. 3. (A) Pallial neuronal densities are significantly higher in parrots and songbirds than in mammals [slopes: F (4,41) = 5.948, P = 0.0007; intercepts: F (4,41) = 75.688, P = < 10−6; post hoc analyses indicate that the regression line for rodents has a different slope and that parrots and songbirds have significantly higher telencephalic neuronal densities than primates and artiodactyls, P < 10−6 for all planned comparisons]. (B) Cerebellar neuronal densities tend to be higher in parrots and songbirds than in mammals [slopes: F (4,40) = 7.84, P < 10−4; intercepts: F (4,40) = 24.71, P = < 10−6; post hoc analyses indicate that the regression line for primates has a different slope and that parrots and songbirds have significantly higher cerebellar neuronal densities than rodents and artiodactyls, P < 10−4 for all planned comparisons]. (C) Neuronal densities in the rest of brain are significantly higher in parrots and songbirds than in mammals [slopes: F (4,41) = 4.876, P = 0.003; intercepts: F (4,41) = 86.875, P = < 10−6; post hoc analyses indicate that the regression line for parrots and for rodents differ in slope from other regression lines and that songbirds have significantly higher neuronal densities than primates and artiodactyls, P < 10−6 for all planned comparisons]. Ad Fig. 5. (C) Allometric lines for songbirds (green line) and parrots (red line) differ significantly in slope [F (1,20) = 17.232, P = 0.0005].

Acknowledgments We thank O. Güntürkün, H. J. ten Donkelaar, T. Bugnyar, N. C. Bennett, M. Prevorovsky, and K. Kverkova for reading of the manuscript and discussions; V. Miller and T. Hajek for logistic support; Y. Zhang and V. Blahova for their assistance with experiments; Z. Pavelkova and B. Strakova for collecting data on avian and mammalian brain and body mass from the literature; L. Kratochvil for methodological advice; P. Benda and J. Mateju for help with acquiring animal experiment approvals; R. Vodicka for assistance with anaesthesia of the emu; and P. Benda and J. Mlikovsky for providing access to dissection facilities of the National Museum of the Czech Republic. This project was funded by Czech Science Foundation (14-21758S) (to P.N.), Grant Agency of Charles University (851613) (to M.K.), Specific Research Grant from Charles University in Prague (SVV 260 313/2016) (to M.K.), the European Social Fund and the state budget of the Czech Republic (CZ.1.07/2.3.00/30.0022) (to S.O.)., the Brazilian National Council for Scientific and Technological Development (to S.H.-H.), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (to S.H.-H.), and the James S. McDonnell Foundation (to S.H.-H.).

Footnotes Author contributions: S.O., M.K., S.H.-H., and P.N. designed research; S.O., M.K., R.K.L., M.P., and P.N. performed research; R.K.L. and M.P. collected experimental animals; S.O., M.K., S.H.-H., and P.N. analyzed data; and S.O., M.K., W.T.F., S.H.-H., and P.N. wrote the paper.

The authors declare no conflict of interest.

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