Vincent Van Meir

In vivo manganese-enhanced magnetic resonance imaging reveals connections and functional properties of the songbird vocal control system

Injection of manganese (Mn(2+)), a paramagnetic tract tracing agent and calcium analogue, into the high vocal center of starlings labeled within a few hours the nucleus robustus archistriatalis and area X as observed by in vivo magnetic resonance imaging. Structures highlighted by Mn(2+) accumulation assumed the expected tri-dimensional shape of the nucleus robustus archistriatalis and area X as identified by classical histological or neurochemical methods. The volume of these nuclei could be accurately calculated by segmentation of the areas highlighted by Mn(2+). Besides confirming previously established volumetric sex differences, Mn(2+) uptake into these nuclei revealed new functional sex differences affecting Mn(2+) transport. A faster transport was observed in males than in females and different relative amounts of Mn(2+) were transported to nucleus robustus archistriatalis and area X in males as compared to females. This new in vivo approach, allowing repeated measures, opens new vistas to study the remarkable seasonal plasticity in size and activity of song-control nuclei and correlate neuronal activity with behavior. It also provides new insights on in vivo axonal transport and neuronal activity in song-control nuclei of oscines.

A three-dimensional MRI atlas of the zebra finch brain in stereotaxic coordinates.

The neurobiology of birdsong, as a model for human speech, is a fast growing area of research in the neurosciences and involves electrophysiological, histological and more recently magnetic resonance imaging (MRI) approaches. Many of these studies require the identification and localization of different brain areas (nuclei) involved in the sensory and motor control of song. Until now, the only published atlases of songbird brains consisted in drawings based on histological slices of the canary and of the zebra finch brain. Taking advantage of high-magnetic field (7 Tesla) MRI technique, we present the first high-resolution (80 x 160 x 160 microm) 3-D digital atlas in stereotaxic coordinates of a male zebra finch brain, the most widely used species in the study of birdsong neurobiology. Image quality allowed us to discern most of the song control, auditory and visual nuclei. The atlas can be freely downloaded from our Web site and can be interactively explored with MRIcro. This zebra finch MRI atlas should become a very useful tool for neuroscientists working on birdsong, especially for co-registrating MRI data but also for determining accurately the optimal coordinates and angular approach for injections or electrophysiological recordings.

Differential effects of testosterone on neuronal populations and their connections in a sensorimotor brain nucleus controlling song production in songbirds: a manganese enhanced-magnetic resonance imaging study

Nucleus HVC (formerly called high vocal center) of songbirds contains two types of projecting neurons connecting HVC respectively to the nucleus robustus archistriatalis, RA, or to area X. These two neuron classes exhibit multiple neurochemical differences and are differentially replaced by new neurons during adult life: high rates of neuronal replacement are observed in RA-projecting neurons only. The activity of these two types of neurons may also be modulated differentially by steroids. We analyzed by magnetic resonance imaging the effect of testosterone on the volume of RA and area X and on the dynamics of Mn(2+) accumulation in RA and area X of female starlings that had been injected with MnCl(2) through a permanent cannula implanted in HVC. Repeated visualization 6 weeks apart (before and after testosterone treatment) identified a volume increase of both nuclei in testosterone-treated birds associated with a concomitant decrease in controls. Following testosterone treatment, the total amount of Mn(2+) transported to RA and area X increased but the dynamics of accumulation, reflecting in part the activity of HVC neurons, was specifically altered in area X but not in RA. These data indicate that testosterone differentially affects the RA- and area X-projecting neurons in HVC. Manganese-enhanced magnetic resonance imaging (ME-MRI) thus provides repeated measures of connected brain areas and demonstrates testosterone-dependent regionally specific changes in brain activity and functional connectivity. The slow time scales investigated by this technique (compared to functional MRI) appear ideally suited for characterizing slow processes such as those involved in brain plasticity and learning.

MRI in small brains displaying extensive plasticity

Manganese-enhanced magnetic resonance imaging (ME-MRI), blood oxygen-level-dependent functional MRI (BOLD fMRI) and diffusion tensor imaging (DTI) can now be applied to animal species as small as mice or songbirds. These techniques confirmed previous findings but are also beginning to reveal new phenomena that were difficult or impossible to study previously. These imaging techniques will lead to major technical and conceptual advances in systems neurosciences. We illustrate these new developments with studies of the song control and auditory systems in songbirds, a spatially organized neuronal circuitry that mediates the acquisition, production and perception of complex learned vocalizations. This neural system is an outstanding model for studying vocal learning, brain steroid hormone action, brain plasticity and lateralization of brain function.

Seasonal rewiring of the songbird brain: an in vivo MRI study.

The song control system (SCS) of songbirds displays a remarkable plasticity in species where song output changes seasonally. The mechanisms underlying this plasticity are barely understood and research has primarily been focused on the song nuclei themselves, largely neglecting their interconnections and connections with other brain regions. We investigated seasonal changes in the entire brain, including the song nuclei and their connections, of nine male starlings (Sturnus vulgaris). At two times of the year, during the breeding (April) and nonbreeding (July) seasons, we measured in the same subjects cellular attributes of brain regions using in vivo high‐resolution diffusion tensor imaging (DTI) at 7 T. An increased fractional anisotropy in the HVC–RA pathway that correlates with an increase in axonal density (and myelination) was found during the breeding season, confirming multiple previous histological reports. Other parts of the SCS, namely the occipitomesencephalic axonal pathway, which contains fiber tracts important for song production, showed increased fractional anisotropy due to myelination during the breeding season and the connection between HVC and Area X showed an increase in axonal connectivity. Beyond the SCS we discerned fractional anisotropy changes that correlate with myelination changes in the optic chiasm and axonal organization changes in an interhemispheric connection, the posterior commissure. These results demonstrate an unexpectedly broad plasticity in the connectivity of the avian brain that might be involved in preparing subjects for the competitive and demanding behavioral tasks that are associated with successful reproduction.

In vivo MR imaging of the seasonal volumetric and functional plasticity of song control nuclei in relation to song output in a female songbird.

In temperate zone songbird species, seasonal plasticity in the morphological and functional state of brain regions involved in song production occurs in association with seasonal changes in song output. Following MnCl(2)-injections in HVC (used as proper name) of female starlings, in vivo tract-tracing by Manganese Enhanced-Magnetic Resonance Imaging (ME-MRI) provided repeated measures of the volume of two HVC targets, the nucleus robustus arcopallii (RA) and area X, along with measures of the activity of the caudal motor pathway and rostral basal-ganglia pathway that control singing. Mn(2+)-labeling (volume labeled and signal intensity) of both nuclei was dramatically reduced in July (post-breeding season) when birds did not sing, compared to March (breeding season) when birds produced song. Seasonal changes in telencephalon volume did not exceed 4% and were not significant but were surprisingly correlated with individual measures of song rate and song bout length. Although individual song rates were variable in March, all MnCl(2)-injections led to a reliable labeling of area X and RA. In July, delineation of area X was only possible in two birds and RA could be delineated in 50% of the population; its volume had decreased by 46% as compared to March. The birds in which RA could be delineated in July had in March a higher activity of the HVC to area X projection as reflected by the total amount of Mn(2+) accumulated in area X, which suggests unexpected relationships between the two types of HVC projection neurons.

In vivo diffusion tensor imaging (DTI) of brain subdivisions and vocal pathways in songbirds.

The neural substrate for song behavior in songbirds, the song control system (SCS), is thus far the best-documented brain circuit in which to study neuroplasticity and adult neurogenesis. Not only does the volume of the key song control nuclei change in size, but also the density of the connections between them changes as a function of seasonal and hormonal influences. This study explores the potentials of in vivo Diffusion-Tensor MRI (DT-MRI or DTI) to visualize the distinct, concentrated connections of the SCS in the brain of the starling (Sturnus vulgaris). In vivo DTI on starling was performed on a 7T MR system using sagittal and coronal slices. DTI was accomplished with diffusion gradients applied in seven non-collinear directions. Fractional Anisotropy (FA)-maps allowed us to distinguish most of the grey matter and white matter-tracts, including the laminae subdividing the avian telencephalon and the tracts connecting the major song control nuclei (e.g., HVC with RA and X). The FA-maps also allowed us to discern a number of song control, auditory and visual nuclei. Fiber tracking was implemented to illustrate the discrimination of all tracts running from and to RA. Because of the remarkable plasticity inherent to the songbird brain, the successful implementation of DTI in this model could represent a useful tool for the in vivo exploration of fiber degeneration and regeneration and the biological mechanisms involved in brain plasticity.

General and efficient super-resolution method for multi- slice MRI

In this paper, a method is developed that reconstructs a high resolution image from an arbitrary set of multi-slice 3D MR images with a high in-plane resolution and a low through-plane resolution. Such images are often recorded to increase the efficiency of the acquisition. With a model of the acquisition of MR images, which is improved compared to previous super-resolution methods for MR images, a large system with linear equations is obtained. With the conjugated gradient method and this linear system, a high resolution image is reconstructed from MR images of an object. Also, a new and efficient method to apply an affine transformation to multi-dimensional images is presented. This method is used to efficiently reconstruction the high resolution image from multislice MR images with arbitrary orientations of the slices.

A customizable 3-dimensional digital atlas of the canary brain in multiple modalities

Songbirds are well known for their ability to learn their vocalizations by imitating conspecific adults. This uncommon skill has led to many studies examining the behavioral and neurobiological processes involved in vocal learning. Canaries display a variable, seasonally dependent, vocal behavior throughout their lives. This trait makes this bird species particularly valuable to study the functional relationship between the continued plasticity in the singing behavior and alterations in the anatomy and physiology of the brain. In order to optimally interpret these types of studies, a detailed understanding of the brain anatomy is essential. Because traditional 2-dimensional brain atlases are limited in the information they can provide about the anatomy of the brain, here we present a 3-dimensional MRI-based atlas of the canary brain. Using multiple imaging protocols we were able to maximize the number of detectable brain regions, including most of the areas involved in song perception, learning, and production. The brain atlas can readily be used to determine the stereotactic location of delineated brain areas at any desirable head angle. Alternatively the brain data can be used to determine the ideal orientation of the brain for stereotactic injections, electrophysiological recordings, and brain sectioning. The 3-dimensional canary brain atlas presented here is freely available and is easily adaptable to support many types of neurobiological studies, including anatomical, electrophysiological, histological, explant, and tracer studies.

Does similarity in call structure or foraging ecology explain interspecific information transfer in wild Myotis bats

Animals can gain important information by attending to the signals and cues of other animals in their environment, with acoustic information playing a major role in many taxa. Echolocation call sequences of bats contain information about the identity and behaviour of the sender which is perceptible to close-by receivers. Increasing evidence supports the communicative function of echolocation within species, yet data about its role for interspecific information transfer is scarce. Here, we asked which information bats extract from heterospecific echolocation calls during foraging. In three linked playback experiments, we tested in the flight room and field if foraging Myotis bats approached the foraging call sequences of conspecifics and four heterospecifics that were similar in acoustic call structure only (acoustic similarity hypothesis), in foraging ecology only (foraging similarity hypothesis), both, or none. Compared to the natural prey capture rate of 1.3 buzzes per minute of bat activity, our playbacks of foraging sequences with 23–40 buzzes/min simulated foraging patches with significantly higher profitability. In the flight room, M. capaccinii only approached call sequences of conspecifics and of the heterospecific M. daubentonii with similar acoustics and foraging ecology. In the field, M. capaccinii and M. daubentonii only showed a weak positive response to those two species. Our results confirm information transfer across species boundaries and highlight the importance of context on the studied behaviour, but cannot resolve whether information transfer in trawling Myotis is based on acoustic similarity only or on a combination of similarity in acoustics and foraging ecology.Significance statementAnimals transfer information, both voluntarily and inadvertently, and within and across species boundaries. In echolocating bats, acoustic call structure and foraging ecology are linked, making echolocation calls a rich source of information about species identity, ecology and activity of the sender, which receivers might exploit to find profitable foraging grounds. We tested in three lab and field experiments if information transfer occurs between bat species and if bats obtain information about ecology from echolocation calls. Myotis capaccinii/daubentonii bats approached call playbacks, but only those from con- and heterospecifics with similar call structure and foraging ecology, confirming interspecific information transfer. Reactions differed between lab and field, emphasising situation-dependent differences in animal behaviour, the importance of field research, and the need for further studies on the underlying mechanism of information transfer and the relative contributions of acoustic and ecological similarity.