The role of MRI in applying the 3Rs to non-human primate neuroscience.

Magnetic resonance imaging is playing a significant role in applying the 3Rs to neuroscience studies using non-human primates. MRI scans are contributing to refinement by enhancing the selection and assignment of animals, guiding the manufacture of custom-fitted recording and head fixation devices, and assisting with the diagnosis of health issues and their treatment. MRI is also being used to better understand the impact of neuroscience procedures on the welfare of NHPs. MRI has helped to optimise NHP use and make greater scientific progress than would otherwise be made using larger numbers of animals. Whilst human fMRI studies have replaced some NHP studies, their potential to directly replace NHP electrophysiology is limited at present. Given the considerable advantages of MRI for electrophysiology experiments, including improved welfare of NHPs, consideration should be given to focusing NHP electrophysiology laboratories around MRI facilities. Greater sharing of MRI data sets, and improvements in MRI contrast and resolution, are expected to further advance the 3Rs in the future.

International primate neuroscience research regulation, public engagement and transparency opportunities.

Scientific excellence is a necessity for progress in biomedical research. As research becomes ever more international, establishing international collaborations will be key to advancing our scientific knowledge. Understanding the similarities in standards applied by different nations to animal research, and where the differences might lie, is crucial. Cultural differences and societal values will also contribute to these similarities and differences between countries and continents. Our overview is not comprehensive for all species, but rather focuses on non-human primate (NHP) research, involving New World marmosets and Old World macaques, conducted in countries where NHPs are involved in neuroimaging research. Here, an overview of the ethics and regulations is provided to help assess welfare standards amongst primate research institutions. A comparative examination of these standards was conducted to provide a basis for establishing a common set of standards for animal welfare. These criteria may serve to develop international guidelines, which can be managed by an International Animal Welfare and Use Committee (IAWUC). Internationally, scientists have a moral responsibility to ensure excellent care and welfare of their animals, which in turn, influences the quality of their research. When working with animal models, maintaining a high quality of care ("culture of care") and welfare is essential. The transparent promotion of this level of care and welfare, along with the results of the research and its impact, may reduce public concerns associated with animal experiments in neuroscience research.

Beyond MRI: on the scientific value of combining non-human primate neuroimaging with metadata.

Sharing and pooling large amounts of non-human primate neuroimaging data offer new exciting opportunities to understand the primate brain. The potential of big data in non-human primate neuroimaging could however be tremendously enhanced by combining such neuroimaging data with other types of information. Here we describe metadata that have been identified as particularly valuable by the non-human primate neuroimaging community, including behavioural, genetic, physiological and phylogenetic data.

Strengths and challenges of longitudinal non-human primate neuroimaging.

Longitudinal non-human primate neuroimaging has the potential to greatly enhance our understanding of primate brain structure and function. Here we describe its specific strengths, compared to both cross-sectional non-human primate neuroimaging and longitudinal human neuroimaging, but also its associated challenges. We elaborate on factors guiding the use of different analytical tools, subject-specific versus age-specific templates for analyses, and issues related to statistical power.

Auditory motion-specific mechanisms in the primate brain.

This work examined the mechanisms underlying auditory motion processing in the auditory cortex of awake monkeys using functional magnetic resonance imaging (fMRI). We tested to what extent auditory motion analysis can be explained by the linear combination of static spatial mechanisms, spectrotemporal processes, and their interaction. We found that the posterior auditory cortex, including A1 and the surrounding caudal belt and parabelt, is involved in auditory motion analysis. Static spatial and spectrotemporal processes were able to fully explain motion-induced activation in most parts of the auditory cortex, including A1, but not in circumscribed regions of the posterior belt and parabelt cortex. We show that in these regions motion-specific processes contribute to the activation, providing the first demonstration that auditory motion is not simply deduced from changes in static spatial location. These results demonstrate that parallel mechanisms for motion and static spatial analysis coexist within the auditory dorsal stream.

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.

Own-song recognition in the songbird auditory pathway: selectivity and lateralization.

The songbird brain is able to discriminate between the bird's own song and other conspecific songs. Determining where in the brain own- song selectivity emerges is of great importance because experience-dependent mechanisms are necessarily involved and because brain regions sensitive to self-generated vocalizations could mediate auditory feedback that is necessary for song learning and maintenance. Using functional MRI, here we show that this selectivity is present at the midbrain level. Surprisingly, the selectivity was found to be lateralized toward the right side, a finding reminiscent of the potential right lateralization of song production in zebra finches but also of own-face and own-voice recognition in human beings. These results indicate that a midbrain structure can process subtle information about the identity of a subject through experience-dependent mechanisms, challenging the classical perception of subcortical regions as primitive and nonplastic structures. They also open questions about the evolution of the cognitive skills and lateralization in vertebrates.

Structural changes between seasons in the songbird auditory forebrain.

The song control system (SCS) of seasonal songbirds shows remarkable seasonal plasticity. Male starlings (Sturnus vulgaris) sing throughout the year, but in the breeding season, when concentrations of testosterone are elevated, the song is highly sexually motivated. The main goal of this study was to investigate structural seasonal changes in regions involved in auditory processing and in socio-sexual behavior. Using in vivo Diffusion Tensor Imaging (DTI), we measured in breeding and nonbreeding seasons volume and tissue characteristics of several brain regions of nine adult male starlings. We demonstrate that the songbird brain exhibits an extreme seasonal plasticity not merely limited to the SCS. Volumetric analysis showed seasonal telencephalon volume changes and more importantly also a volumetric change in the caudal region of the nidopallium (NCM), a region analogous to the mammalian secondary auditory cortex. Analysis of the DTI data allowed detection of seasonal changes in cellular attributes in NCM and regions involved in social behavior. This study extends our view on a seasonally dynamic avian brain which not only hones its song control system but also auditory and social systems to be prepared for the breeding season.

Implementation of spin-echo blood oxygen level-dependent (BOLD) functional MRI in birds.

The advent of high-field MRI systems has allowed the implementation of blood oxygen level-dependent functional MRI (BOLD fMRI) on small animals. An increased magnetic field improves the signal-to-noise ratio and thus allows an improvement in the spatial resolution. However, it also increases susceptibility artefacts in the commonly acquired gradient-echo images. This problem is particularly prominent in songbird MRI because of the presence of numerous air cavities in the skull of birds. These T(2)*-related image artefacts can be circumvented using spin-echo BOLD fMRI. In this article, we describe the implementation of spin-echo BOLD fMRI in zebra finches, a small songbird of 15-25 g, extensively studied in the behavioural neurosciences of birdsong. Because the main topics in this research domain are song perception and song learning, the protocol implemented used auditory stimuli. Despite the auditory nature of the stimuli and the weak contrast-to-noise ratio of spin-echo BOLD fMRI compared with gradient-echo BOLD fMRI, we succeeded in detecting statistically significant differences in BOLD responses triggered by different stimuli. This study shows that spin-echo BOLD fMRI is a viable approach for the investigation of auditory processing in the whole brain of small songbirds. It can also be applied to study auditory processing in other small animals, as well as other sensory modalities.

Pacing behaviour in laboratory macaques is an unreliable indicator of acute stress.

Pacing behaviour, the most frequent stereotypic behaviour displayed by laboratory rhesus macaques (Macaca mulatta) is often used as an indicator of stress. In this study, we investigated how reliable this welfare indicator is at detecting acute stress by testing the reaction of macaques to the stressful event of being exposed to an agonistic interaction between conspecifics housed in the same room but in a different cage. Pacing, agitated locomotion, and stress-related displacement behaviours were quantified before, during and after agonistic interaction exposure, based on video recordings of 13 socially-housed macaques in their home cage. Displacement behaviours increased after agonistic interaction exposure, confirming that the events were experienced as stressful by the focal individuals. The occurrence of pacing did not increase during or after the agonistic interactions. Instead, agitated locomotion increased during the agonistic interactions. These results suggest either, that pacing as an indicator of acute stress is prone to false negative results, increasing in some stressful situations but not others, or that agitated locomotion has been mistaken for pacing in previous studies and that pacing is in fact unrelated to current acute stress. Both interpretations lead to the conclusion that pacing is unreliable as an indicator of acute stress in laboratory rhesus macaques.

Validation of hippocampal biomarkers of cumulative affective experience.

Progress in improving the welfare of captive animals has been hindered by a lack of objective indicators to assess the quality of lifetime experience, often called cumulative affective experience. Recent developments in stress biology and psychiatry have shed new light on the role of the mammalian hippocampus in affective processes. Here we review these findings and argue that structural hippocampal biomarkers demonstrate criterion, construct and content validity as indicators of cumulative affective experience in mammals. We also briefly review emerging findings in birds and fish, which have promising implications for applying the hippocampal approach to these taxa, but require further validation. We hope that this review will motivate welfare researchers and neuroscientists to explore the potential of hippocampal biomarkers of cumulative affective experience.

An Open Resource for Non-human Primate Imaging.

Non-human primate neuroimaging is a rapidly growing area of research that promises to transform and scale translational and cross-species comparative neuroscience. Unfortunately, the technological and methodological advances of the past two decades have outpaced the accrual of data, which is particularly challenging given the relatively few centers that have the necessary facilities and capabilities. The PRIMatE Data Exchange (PRIME-DE) addresses this challenge by aggregating independently acquired non-human primate magnetic resonance imaging (MRI) datasets and openly sharing them via the International Neuroimaging Data-sharing Initiative (INDI). Here, we present the rationale, design, and procedures for the PRIME-DE consortium, as well as the initial release, consisting of 25 independent data collections aggregated across 22 sites (total = 217 non-human primates). We also outline the unique pitfalls and challenges that should be considered in the analysis of non-human primate MRI datasets, including providing automated quality assessment of the contributed datasets.

What neuroimaging tells us about sensory substitution.

A major question in the field of sensory substitution concerns the nature of the perception generated by sensory substitution prostheses. Is the perception determined by the nature of the substitutive modality or is it determined by the nature of the information transmitted by the device? Is it a totally new, amodal, perception? This paper reviews the recent neuroimaging studies which have investigated the neural bases of sensory substitution. The detailed analysis of available results led us to propose a general scheme of the neural mechanisms underlying sensory substitution. Two different main processes may be responsible for the visual area recruitment observed in the different studies: cross-modality and mental (visual) imagery. Based on our results analysis, we propose that cross-modality is the predominant process in early blind subjects whereas mental imagery is predominant in blindfolded sighted subjects. This model implies that, with training, sensory substitution mainly induces visual-like perception in sighted subjects and mainly auditory or tactile perception in blind subjects. This framework leads us to make some predictions that could easily be tested.

Pacing stereotypies in laboratory rhesus macaques: Implications for animal welfare and the validity of neuroscientific findings.

Stereotypic behaviours are commonly observed in captive animals and are usually interpreted as a sign of poor welfare. Stereotypies have also been linked with brain abnormalities. However, stereotypies are a heterogeneous class of behaviours and mounting evidence indicates that different stereotypies can have different causes, and can be linked to different affective states. As a consequence, the implications of a specific stereotypy in a specific species cannot be safely inferred from evidence on other stereotypies or species. Here we review what is known about pacing behaviour in laboratory rhesus macaques, a common stereotypy in this species. Our review highlights the current lack of understanding of the causal factors underlying pacing behaviour. According to current knowledge, the welfare of pacing macaques could be either better, worse or equivalent to that of non-pacing individuals. It is also unclear whether pacing results from brain abnormalities. Since rhesus macaques are widely used as a model of healthy humans in neuroscience research, determining if pacing behaviour reflects an abnormal brain and/or poor welfare is urgent.

Specific activation of the V5 brain area by auditory motion processing: an fMRI study.

Previous neuroimaging studies devoted to auditory motion processing have shown the involvement of a cerebral network encompassing the temporoparietal and premotor areas. Most of these studies were based on a comparison between moving stimuli and static stimuli placed at a single location. However, moving stimuli vary in spatial location, and therefore motion detection can include both spatial localisation and motion processing. In this study, we used fMRI to compare neural processing of moving sounds and static sounds in various spatial locations in blindfolded sighted subjects. The task consisted of simultaneously determining both the nature of a sound stimulus (pure tone or complex sound) and the presence or absence of its movement. When movement was present, subjects had to identify its direction. This comparison of how moving and static stimuli are processed showed the involvement of the parietal lobules, the dorsal and ventral premotor cortex and the planum temporale during auditory motion processing. It also showed the specific recruitment of V5, the visual motion area. These results suggest that the previously proposed network of auditory motion processing is distinct from the network of auditory localisation. In addition, they suggest that the occipital cortex can process non-visual stimuli and that V5 is not restricted to visual processing.

Direct social contacts override auditory information in the song-learning process in starlings (Sturnus vulgaris).

Social influence on song acquisition was studied in 3 groups of young European starlings raised under different social conditions but with the same auditory experience of adult song. Attentional focusing on preferred partners appears the most likely explanation for differences found in song acquisition in relation to experience, sex, and song categories. Thus, pair-isolated birds learned from each other and not from broadcast live songs, females did not learn from the adult male tutors, and sharing occurred more between socially associated peers. On the contrary, single-isolated birds clearly copied the adult songs that may have been the only source of attention stimulation. Therefore, social preference appears as both a motor for song learning and a potential obstacle for acquisition from nonpreferred partners, including adults.

A perceptual pitch boundary in a non-human primate.

Pitch is an auditory percept critical to the perception of music and speech, and for these harmonic sounds, pitch is closely related to the repetition rate of the acoustic wave. This paper reports a test of the assumption that non-human primates and especially rhesus monkeys perceive the pitch of these harmonic sounds much as humans do. A new procedure was developed to train macaques to discriminate the pitch of harmonic sounds and thereby demonstrate that the lower limit for pitch perception in macaques is close to 30 Hz, as it is in humans. Moreover, when the phases of successive harmonics are alternated to cause a pseudo-doubling of the repetition rate, the lower pitch boundary in macaques decreases substantially, as it does in humans. The results suggest that both species use neural firing times to discriminate pitch, at least for sounds with relatively low repetition rates.

Comparisons of different methods to train a young zebra finch (Taeniopygia guttata) to learn a song.

Like humans, oscine songbirds exhibit vocal learning. They learn their song by imitating conspecifics, mainly adults. Among them, the zebra finch (Taeniopygia guttata) has been widely used as a model species to study the behavioral, cellular and molecular substrates of vocal learning. Various methods using taped song playback have been used in the laboratory to train young male finches to learn a song. Since different protocols have been applied by different research groups, the efficiency of the studies cannot be directly compared. The purpose of our study was to address this problem. Young finches were raised by their mother alone from day post hatching (dph) 10 and singly isolated from dph 35. One week later, exposure to a song model began, either using a live tutor or taped playback (passive or self-elicited). At dph 100, the birds were transferred to a common aviary. We observed that one-to-one live tutoring is the best method to get a fairly complete imitation. Using self-elicited playback we observed high inter-individual variability; while some finches learned well (including good copying of the song model), others exhibited poor copying. Passive playback resulted in poor imitation of the model. We also observed that finches exhibited vocal changes after dph 100 and that the range of these changes was negatively related to their imitation of the song model. Taken together, these results suggest that social aspects are predominant in the success outcome of song learning in the zebra finch.

Representation of early sensory experience in the adult auditory midbrain: implications for vocal learning.

Vocal learning in songbirds and humans occurs by imitation of adult vocalizations. In both groups, vocal learning includes a perceptual phase during which juveniles birds and infants memorize adult vocalizations. Despite intensive research, the neural mechanisms supporting this auditory memory are still poorly understood. The present functional MRI study demonstrates that in adult zebra finches, the right auditory midbrain nucleus responds selectively to the copied vocalizations. The selective signal is distinct from selectivity for the bird's own song and does not simply reflect acoustic differences between the stimuli. Furthermore, the amplitude of the selective signal is positively correlated with the strength of vocal learning, measured by the amount of song that experimental birds copied from the adult model. These results indicate that early sensory experience can generate a long-lasting memory trace in the auditory midbrain of songbirds that may support song learning.

Functional changes between seasons in the male songbird auditory forebrain.

Songbirds are an excellent model for investigating the perception of learned complex acoustic communication signals. Male European starlings (Sturnus vulgaris) sing throughout the year distinct types of song that bear either social or individual information. Although the relative importance of social and individual information changes seasonally, evidence of functional seasonal changes in neural response to these songs remains elusive. We thus decided to use in vivo functional magnetic resonance imaging (fMRI) to examine auditory responses of male starlings that were exposed to songs that convey different levels of information (species-specific and group identity or individual identity), both during (when mate recognition is particularly important) and outside the breeding season (when group recognition is particularly important). We report three main findings: (1) the auditory area caudomedial nidopallium (NCM), an auditory region that is analogous to the mammalian auditory cortex, is clearly involved in the processing/categorization of conspecific songs; (2) season-related change in differential song processing is limited to a caudal part of NCM; in the more rostral parts, songs bearing individual information induce higher BOLD responses than songs bearing species and group information, regardless of the season; (3) the differentiation between songs bearing species and group information and songs bearing individual information seems to be biased toward the right hemisphere. This study provides evidence that auditory processing of behaviorally-relevant (conspecific) communication signals changes seasonally, even when the spectro-temporal properties of these signals do not change.