Failure to detect seasonal changes in the song system nuclei of the black-capped chickadee (Poecile atricapillus).

Most temperate songbird species sing seasonally, and the brain areas involved in producing song (the song system) vary in size alongside the changes in behavior. Black-capped chickadees (Poecile atricapillus) also sing seasonally, and we find that there are changes in the stereotypy and the length of the fee-bee song from the nonbreeding to the breeding season. Yet despite these changes, we fail to find any evidence of seasonal changes in the song system. The song system of males is larger than that of females, as is typical in songbirds, but the ratio between the sexes is small compared to other species. We suggest three hypotheses to explain our failure to find seasonal variation in the chickadee song system.

The distribution of substance P and neuropeptide Y in four songbird species: a comparison of food-storing and non-storing birds.

The distributions of the neuropeptides substance P (SP) and neuropeptide Y (NPY) were investigated in four songbird species that differ in their food-storing behavior. The food-storing black-capped chickadee (Parus atricapillus) was compared to the non-storing blue tit (Parus caeruleus) and great tit (Parus major) within the avian family Paridae, as well as to the non-storing dark-eyed junco (Junco hyemalis). All four species showed a similar distribution of SP throughout the brain with the exception of two areas, the hippocampal complex (including hippocampus (Hp) and parahippocampus (APH)) and the Wulst (including the hyperstriatum accessorium (HA)). SP-like immunoreactivity was found in cells of the Hp in juncos, but not in the three parid species. Two areas within the APH and HA showed SP-like immunoreactivity in all four species. The more medial of these (designated SPm) is a distinctive field of fibers and terminals found throughout the APH and extending into the HA. A positive relationship between SPm and Hp volume was found for all four species with the chickadee having a significantly larger SPm area relative to telencephalon than the other species. The distribution of SP in this region may be related to differences in food-storing behavior. In contrast to substance P, NPY distribution throughout the brain was similar in all four species. Further, NPY-immunoreactive cells were found in the Hp of all four species and no species differences in the number of NPY cells was observed.

Expression of immediate early genes in the hippocampal formation of the black-capped chickadee (Poecile atricapillus) during a food-hoarding task.

Black-capped chickadees store food in many different locations in their home range and are able to accurately remember these locations. We measured the number of cells immunopositive for three different Immediate Early Gene products (Fra-1, c-Fos and ZENK) to map neuronal activity in the chickadee Hippocampal Formation (HF) during food storing and retrieval. Fra-1-like immunoreactivity is downregulated in the dorsal HF of both storing and retrieving chickadees compared to controls. In retrieving birds, the number of Fos-like immunoreactive neurons relates to the number of items remembered, while the number of ZENK-like immunoreactive neurons in the HF may be related to the accuracy of cache retrieval. These results imply that the brain might process complex information by recruiting more neurons into the network of active neurons. Thus, our results could help explain why food-hoarding birds have more HF neurons than non-hoarders, and why this number increases in autumn when large numbers of food items are cached.

Seasonal changes in neuron numbers in the hippocampal formation of a food-hoarding bird: the black-capped chickadee.

The volume of the hippocampal formation (HF) in black-capped chickadees (Poecile atricapillus) varies across the seasons, in parallel with the seasonal cycle in food hoarding. In this study, we estimate cell density and total cell number in the HF across seasons in both juveniles and adults. We find that the seasonal variation in volume is due to an increase in the number of small and large cells (principally neurons) in the fall. Adults also have lower neuron densities than juveniles. Both juveniles and adults show an increase in cell density in the rostral part of the HF in August and a subsequent decrease toward October. This suggests that the net cell addition to the HF may already start in August. We discuss the implications of this early start with respect to the possibility that the seasonal change in HF volume is driven by the experience of food hoarding. We also speculate on the functional significance of the addition of neurons to the HF in the fall.

Greater song complexity is associated with augmented song system anatomy in zebra finches.

We revisited the relationship between brain anatomy and song behavior in zebra finches. Consistent with previous studies in other songbirds, we find that differences in volume of the telencephalic song control nucleus HVc is predictive of differences in repertoire size and phrase duration in zebra finches. We extend the study of brain and behavior correlations in song birds by showing that repertoire size in zebra finches can be predicted by variance in several brain nuclei, providing the first demonstration that volumetric differences across multiple components of a neural network are predictive of song behavior.

Song, sexual selection, and a song control nucleus (HVc) in the brains of European sedge warblers.

Female sedge warblers select males that have more complex songs as mates. This study tests two predictions concerning HVc, a telencephalic nucleus that is essential for song learning and production: first, that males with more complex songs will have a larger HVc, and second that males who pair successfully will have a larger HVc than unpaired males. Data on song composition and pairing status were collected from wild sedge warblers breeding in Hungary. We found significant positive correlations between three song attributes (repertoire size, song complexity, and song length) and the size of HVc. Males that paired successfully also had more complex songs (repertoire size and song complexity, though not song length) than males that did not. However, we find no direct evidence that males who paired successfully had a larger HVc than unpaired males. These findings are discussed in relation to the possible functions of HVc and also to current views on sexual selection and the evolution of the song control pathway.

Effects of captivity and testosterone on the volumes of four brain regions in the dark-eyed junco (Junco hyemalis).

This study investigates the effects of captivity and testosterone treatment on the volumes of brain regions involved in processing visual and spatial information in adult dark-eyed juncos (Junco hyemalis). We treated captive and free-living male juncos with either testosterone-filled or empty implants. Captive juncos had a smaller hippocampal formation (HF) (both in absolute volume and relative to telencephalon) than free-living birds, regardless of hormone treatment. Testosterone-treated males (both captive and free-living) had a smaller telencephalon and nucleus rotundus, but not a smaller HF or ectostriatum, than controls. We found that free-living testosterone-treated males had larger home ranges than free-living controls in agreement with earlier experiments, but we found no corresponding difference in HF volume. We discuss the implications of the effect of captivity on HF volume for past and future laboratory experiments.

Greater song complexity is associated with augmented song system anatomy in zebra finches.

We revisited the relationship between brain anatomy and song behavior in zebra finches. Consistent with previous studies in other songbirds, we find that differences in volume of the telencephalic song control nucleus HVc is predictive of differences in repertoire size and phrase duration in zebra finches. We extend the study of brain and behavior correlations in song birds by showing that repertoire size in zebra finches can be predicted by variance in several brain nuclei, providing the first demonstration that volumetric differences across multiple components of a neural network are predictive of song behavior.

Differences in the complexity of song tutoring cause differences in the amount learned and in dendritic spine density in a songbird telencephalic song control nucleus.

In our search for relations between vocal learning and neuron structure in the song control nuclei of songbird forebrains, we tested whether differential experience that leads to differences in adult song repertoire would affect dendritic spine density in HVc (also called high vocal center) and RA (robustus archistriatalis). We tape-tutored juvenile Eastern marsh wrens (Cistothorus palustris) with either 5 or 45 song types. As adults, the small repertoire group had learned mostly 5 or 6 song types, and the large repertoire group had learned 36 to 47. Wrens that learned the large song repertoires had a greater dendritic spine density for the most spiny neurons present in HVc (mean difference, 36%), but not in RA. Recent physiological evidence describes HVc as a premotor area coding syllables, motifs, and higher-order song patterns, and our data now clearly reveal that differences in the size of the song repertoire that is experienced lead to differences both in song learning and in the density of dendritic spines in HVc. In the forebrain song nuclei of these songbirds, as in some other vertebrate systems, differences in learning and performance are associated with differences in synaptic anatomy specifically in the region that organizes the learned pattern.

Variation in the volume of zebra finch song control nuclei is heritable: developmental and evolutionary implications.

In many songbird species, females prefer males that sing a larger repertoire of syllables. Males with more elaborate songs have a larger high vocal centre (HVC) nucleus, the highest structure in the song production pathway. HVC size is thus a potential target of sexual selection. Here we provide evidence that the size of the HVC and other song production nuclei are heritable across individual males within a species. In contrast, we find that heritabilities of other nuclei in a song-learning pathway are lower, suggesting that variation in the sizes of these structures is more closely tied to developmental and environmental differences between individuals. We find that evolvability, a statistical measure that predicts response to selection, is higher for the HVC and its target for song production, the robustus archistriatalis (RA), than for all other brain volumes measured. This suggests that selection based on the functions of these two structures would result in rapid major shifts in their anatomy. We also show that the size of each song control nucleus is significantly correlated with the song related nuclei to which it is monosynaptically connected. Finally, we find that the volume of the telencephalon is larger in males than in females. These findings begin to join theoretical analyses of the role of female choice in the evolution of bird song to neurobiological mechanisms by which the evolutionary changes in behaviour are expressed.

Sexual dimorphism and species differences in HVC volumes of cowbirds.

Cowbirds exhibit extensive variation in their social, territorial, and reproductive behaviors. Nissl-stained brain sections of specimens from a previous study (J. C. Reboreda, N. S. Clayton, & A. Kacelnik, 1996) were used to study the gross anatomy of a song control nucleus in 3 South American cowbirds (bay-winged, Molothrus badius; shiny, M. bonariensis; and screaming, M. rufoaxillaris). Cowbird high vocal center (HVC) volumes were consistently higher in males than in females in all 3 species. The largest HVC size of females found in bay-winged cowbirds is consistent with observations that females of this species, but not of the other 2 species, occasionally sing. The extent of the sexual dimorphism of relative HVC size was highest for the sexually dichromatic and promiscuous shiny cowbirds and smaller for the monochromatic and monogamous bay-winged and screaming cowbirds, suggesting that selection pressures associated with morphological traits and social systems are reflected in brain architecture.

Anterior forebrain pathway is needed for stable song expression in adult male white-crowned sparrows (Zonotrichia leucophrys).

The anterior forebrain pathway of the avian song system is involved in juvenile song learning, but its function in adult song behavior is not known. This report uses lesions to study the role of a particular forebrain nucleus, IMAN, in the seasonal regeneration of song in adult white-crowned sparrows (Zonotrichia leucophrys oriantha). White-crowned sparrows, even when acoustically isolated as juveniles, crystallize a single song which they maintain throughout adulthood. The lateral portion of the magnocellular nucleus of the anterior neostriatum (1MAN) was lesioned bilaterally in adult males maintained on short days (8 h of light). Daylength was increased to 16 h following the surgeries, and all birds were recorded in the post-lesion singing season. Lesioned birds showed a large decrease in song note frequency following the lesions, significantly larger than did intact, age-matched controls. Further changes were seen in the post-lesion songs of seven of 11 successfully lesioned males. These changes included variability in song pattern, loss of frequency control and addition of new notes, some of which had been practiced during juvenile song development. These changes seemed especially large in birds that had either been acoustically isolated or had not fully copied a tape-tutor song during juvenile song development. These results are the first to indicate that the motor memories for song elements that had been practiced and discarded early in life are retained, and they suggest that 1MAN affects seasonal song expression by selectively reinforcing a particular song pattern.

Non invasive in vivo anatomical studies of the oscine brain by high resolution MRI microscopy.

We describe in this paper an in vivo Magnetic Resonance Imaging (MRI) procedure that allows one to obtain three-dimensional high quality images of the entire brain of small passerine birds such as the canary with a slice thickness of 58 micron and an image resolution of 78 microns. This imaging procedure was completed in 70 min on anaesthetised birds that later recovered uneventfully and could be reused for subsequent additional imaging. To illustrate the high resolution and anatomical detail that can be achieved, examples of coronal images through the entire hypothalamus are provided in the same sectioning plane as the previously published canary brain atlas. The data set can be used to create sections in any desired plane and the entire data set can be viewed from any point of view in a volume rendered image. This provides a useful tool in understanding the three-dimensional organisation of the brain. Similar procedures can also be applied on fixed brains and might allow an even better anatomical resolution of images because time constrains no longer limit the duration of image acquisition. The non-invasive MRI technique enables to study neuroanatomical features with a high resolution and without killing the animal subjects so that measures can be obtained in a same individual both before and after an experimental treatment.

Lucifer Yellow filling of area X-projecting neurons in the high vocal center of female canaries.

The avian high vocal center (HVC) is a complex forebrain nucleus that coordinates the sensorimotor integration necessary for song learning and production. It receives auditory and potentially somatosensory input, and sends major projections to vocal motor and anterior forebrain nuclei. The HVC has at least four morphological classes of neurons for which the connectivity remains uncertain. Previous studies have alluded to the functional identity of the cell classes, but none have provided the definitive evidence necessary for subsequent identification of behaviorally relevant changes within known neuronal populations. The cell filling technique we have adapted for use in the song system provides a method by which hodologically identified classes can be described with precision, and song related changes in their morphology can be readily identified. Neurons in female canaries (Serinus canarius) that project to Area X of the anterior forebrain pathway were retrogradely labeled, selectively filled with Lucifer Yellow in a fixed slice preparation, and converted to a Golgi-like stain through an immunocytochemical reaction. We have identified Area X-projecting neurons as belonging to the thick dendrite class of Nixdorf et al. [B.E. Nixdorf, S.S. Davis, T.J. DeVoogd, Morphology of golgi-impregnated neurons in hyperstriatum ventralis, pars caudalis in adult male and female canaries, J. Comp. Neurol. 284 (1989) 337-349] and have shown definitively that they are among the HVC neurons that can receive direct auditory input, as this cell class has short dendrites that extend into the shelf region ventral to HVC that is known to receive auditory inputs. Well-filled axons had collaterals that ramified and terminated within the nucleus, demonstrating a network through which Area X-projecting cells can contribute to intrinsic HVC communication.

White-throated sparrow morphs that differ in song production rate also differ in the anatomy of some song-related brain areas.

White-throated sparrows are unusual among songbirds in that they occur in two color morphs, white-striped and tan-striped, determined by a chromosomal inversion and maintained by negative assortative mating. These differ in several reproductive behaviors, including amount of singing: white-striped males sing frequently, tan-striped females never sing, and tan-striped males and white-striped females sing an intermediate amount. The present study measures the volumes of several nuclei in the avian song system and relates these to color morph and to sex. We find that robustus archistristalis and the tracheosyringeal part of the hypoglossal nucleus, nuclei closely involved in song production, are larger in white-striped than in tan-striped birds. We also find morph differences for nuclei in the rostral division of the song system, nuclei believed to be less directly involved in song production. We find sex differences throughout the song system as has been reported in other songbirds. Relationships between structure and function in the song system are discussed.

Song isolation is associated with maintaining high spine frequencies on zebra finch 1MAN neurons.

Male zebra finches normally learn much of their song during the second month after hatching. This is a period of rapid change throughout the brain. We studied anatomical consequences of manipulating exposure to song. We investigated neurons of lateral MAN (1MAN), a nucleus implicated in song learning (Bottjer et al., 1984), in male and female zebra finches (Taeniopygia guttata) at 55 days posthatch. Birds were raised either under normal colony conditions (social) or in colonies in which adult males were removed when the young hatched (song deprived). Brains were stained with the Golgi-Cox method. Fine morphological details of spiny 1MAN neurons were recorded with a 3D semiautomated computer system. Several features of the spiny 1MAN neurons differ between sexes. Males have neurons with larger somata, more primary dendrites and thicker dendrites, than neurons from females. These features as well as dendritic length and other branching characteristics do not differ between treatment groups. There is a large difference in dendritic spine frequencies between the social and the song-deprived groups. Social, song-experienced males have spine frequencies 41% lower than song-deprived males. In females, spine frequencies are as high as in the song-deprived males and do not differ between the song-deprived and social conditions. Developmental overproduction and subsequent pruning of neural connections have been observed in many areas of the central nervous system. We suggest that apparent pruning in 1MAN is modulated by experience: It takes place if the social experiences associated with auditory song learning have occurred. This finding is consistent with the synapse selection hypothesis of Changeux and Danchin Brain Research, 309, (1976) and with data found using an acoustic filial imprinting paradigm in the domestic chicken.

Developmental changes in the distribution of NADPH-diaphorase-containing neurons in telencephalic nuclei of the zebra finch song system.

Extensive recent research has focused on the potential role of nitric oxide (NO) in synaptic plasticity. Could the capacity to synthesize NO be associated with neural and behavioral plasticity in the song system? The timing of song learning and of major developmental changes in song system anatomy are known. We searched for an association between NO and these developmental events by observing the distribution of neurons staining for NADPH-diaphorase, an enzyme used in the synthesis of NO, in the brains of zebra finches. Both male and female brains were taken at different developmental ages from day 21 to adulthood. We found that the incidence of stained neurons in the song system nuclei is lower than in surrounding areas. The incidence of staining decreases with development, with most of the decrease occurring prior to the auditory learning phase of song learning. The developmental changes were quantified for area X and found to be highly significant, with a 56% decrease in staining frequency from day 21 to adulthood in males and a 23% decrease in females for the equivalent region. We also found a sexual dimorphism in the song system of adult birds, consisting of a reduced incidence of stained neurons in song system nuclei area X, high vocal center (HVC), and nucleus robustus (RA) archistrialis in males compared with females. These findings suggest that NO is less involved in the plasticity underlying song acquisition than in the earlier formation of the song system.

Regressive development in neuronal structure during song learning in birds.

We investigated the development of spiny neurons in the lateral magnocellular nucleus of the anterior neostriatum before, during, and after song learning in male zebra finches (Taeniopygia guttata). The frequency of dendritic spines, dendritic field size, and branching characteristics were quantified at different ages in Golgi-stained tissue using a three-dimensional computerized tracing system. During development, overall spine frequencies increase between 3 and 5 weeks and decrease thereafter. In particular, spine frequencies of middle segments decrease significantly by 14% between 5 and 7 weeks posthatching (p = 0.017). A further reduction of 48% occurs between 7 weeks and adulthood (p < 0.001), resulting in a spine reduction of 56% on middle segments between 35 days of age and adulthood. In addition to the reduction of spine frequencies, we find regressive events also on some of the neuronal parameters that we have quantified. In general, dendrites of adult animals terminate closer to the cell body than those of 7-, 5-, or 3-week-old birds. Whereas no changes in segment length of first- and second-order dendrites have been identified, third-order dendrites end 19% closer to the cell body in adults than in younger birds (p < 0.024). Second-order dendrites in adult animals branch less frequently than in 3-week-old animals (35%, p = 0.017). There is also a trend of a smaller number of tertiary branches in adulthood compared with 3-week-old birds (41%, p = 0.060). The morphological changes may be related to the function of this nucleus and the sensitive phase for song acquisition.

Seasonal variation in hippocampal volume in a food-storing bird, the black-capped chickadee.

Black-capped chickadees (Parus atricapillus) in upstate New York show a peak in food-hoarding intensity in October. We caught chickadees at six different times of the year and measured the volume of several brain structures. We found that the hippocampal formation, which is involved in spatial memory for cached food items, has a larger volume, relative to the rest of the brain, in October than at any other time of the year. We conclude that there is an association between the intensity of food hoarding and the volume of the hippocampal formation and suggest that the enhanced anatomy might be caused by the increased use of spatial memory.