Molecular correlates of hypothalamic development in songbird ontogeny in comparison with the telencephalon.

Development of the songbird brain provides an excellent experimental model for understanding the regulation of sex differences in ontogeny. Considering the regulatory role of the hypothalamus in endocrine, in particular reproductive, physiology, we measured the structural (volume) and molecular correlates of hypothalamic development during ontogeny of male and female zebra finches. We quantified by relative quantitative polymerase chain reaction (rqPCR) the expression of 14 genes related to thyroid and steroid hormones actions as well as 12 genes related to brain plasticity at four specific time points during ontogeny and compared these expression patterns with the expression of the same genes as detected by transcriptomics in the telencephalon. These two different methodological approaches detected specific changes with age and demonstrated that in a substantial number of cases changes observed in both brain regions are nearly identical. Other genes however had a tissue-specific developmental pattern. Sex differences or interactions of sex by age were detected in the expression of a subset of genes, more in hypothalamus than telencephalon. These results correlate with multiple known aspects of the developmental and reproductive physiology but also raise a number of new functional questions.

In Vivo Preclinical Molecular Imaging of Repeated Exposure to an N-methyl-d-aspartate Antagonist and a Glutaminase Inhibitor as Potential Glutamatergic Modulators.

Glutamate is the principal excitatory neurotransmitter in the brain and is at the base of a wide variety of neuropathologies, including epilepsy, autism, Fragile X, and obsessive compulsive disorder. Glutamate has also become the target for novel drugs in treatment and in fundamental research settings. However, much remains unknown on the working mechanisms of these drugs and the effects of chronic administration on the glutamatergic system. This study investigated the chronic effects of two glutamate-modulating drugs with imaging techniques to further clarify their working mechanisms for future research opportunities. Animals were exposed to saline (1 ml/kg), (5S,10R)-(+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) (0.3 mg/kg), or ebselen (10 mg/kg) for 7 consecutive days. At the sixth injection, animals underwent a positron emission tomography (PET)/computed tomography (CT) with (3-(6-methyl-pyridin-2-ylethynyl)-cyclohex-2-enone-O-11C-methyl-oxime) (ABP-688) to visualize the metabotropic G protein-coupled glutamate receptor 5 (mGluR5). After the seventh injection, animals underwent a magnetic resonance spectroscopy (MRS) scan to visualize glutamate and glutamine content. Afterward, results were verified by mGluR5 immunohistochemistry (IHC). PET/CT analysis revealed that animals receiving chronic MK-801 or ebselen had a significant (P < 0.05) higher binding potential (2.90 ± 0.47 and 2.87 ± 0.46, respectively) when compared with saline (1.97 ± 0.39) in the caudate putamen. This was confirmed by mGluR5 IHC, with 60.83% ± 6.30% of the area being highlighted for ebselen and 57.14% ± 9.23% for MK-801 versus 50.21% ± 5.71% for the saline group. MRS displayed significant changes on the glutamine level when comparing chronic ebselen (2.20 ± 0.40 µmol/g) to control (2.72 ± 0.34 µmol/g). Therefore, although no direct effects on glutamate were visualized, the changes in glutamine suggest changes in the total glutamate-glutamine pool. This highlights the potential of both drugs to modulate glutamatergic pathologies.

Neuroplasticity in the cerebello-thalamo-basal ganglia pathway: A longitudinal in vivo MRI study in male songbirds.

Similar to human speech, bird song is controlled by several pathways including a cortico-basal ganglia-thalamo-cortical (C-BG-T-C) loop. Neurotoxic disengagement of the basal ganglia component, i.e. Area X, induces long-term changes in song performance, while most of the lesioned area regenerates within the first months. Importantly however, the timing and spatial extent of structural neuroplastic events potentially affecting other constituents of the C-BG-T-C loop is not clear. We designed a longitudinal MRI study where changes in brain structure were evaluated relative to the time after neurotoxic lesioning or to vocal performance. By acquiring both Diffusion Tensor Imaging and 3-dimensional anatomical scans, we were able to track alterations in respectively intrinsic tissue properties and local volume. Voxel-based statistical analyses revealed structural remodeling remote to the lesion, i.e. in the thalamus and, surprisingly, the cerebellum, both peaking within the first two months after lesioning Area X. Voxel-wise correlations between song performance and MRI parameters uncovered intriguing brain-behavior relationships in several brain areas pertaining to the C-BG-T-C loop supervising vocal motor control. Our results clearly point to structural neuroplasticity in the cerebellum induced by basal ganglia (striatal) damage and might point to the existence of a human-like cerebello-thalamic-basal ganglia pathway capable of modifying vocal motor output.

Rapid changes in auditory processing in songbirds following acute aromatase inhibition as assessed by fMRI.

Contribution to Special Issue on Fast effects of steroids. This review introduces functional MRI (fMRI) as an outstanding tool to assess rapid effects of sex steroids on auditory processing in seasonal songbirds. We emphasize specific advantages of this method as compared to other more conventional and invasive methods used for this purpose and summarize an exemplary auditory fMRI study performed on male starlings exposed to different types of starling song before and immediately after the inhibition of aromatase activity by an i.p. injection of Vorozole™. We describe how most challenges that relate to the necessity to anesthetize subjects and minimize image- and sound-artifacts can be overcome in order to obtain a voxel-based 3D-representation of changes in auditory brain activity to various sound stimuli before and immediately after a pharmacologically-induced depletion of endogenous estrogens. Analysis of the fMRI data by assumption-free statistical methods identified fast specific changes in activity in the auditory brain regions that were stimulus-specific, varying over different seasons, and in several instances lateralized to the left side of the brain. This set of results illustrates the unique features of fMRI that provides opportunities to localize and quantify the brain responses to rapid changes in hormonal status. fMRI offers a new image-guided research strategy in which the spatio-temporal profile of fast neuromodulations can be identified and linked to specific behavioral inputs or outputs. This approach can also be combined with more localized invasive methods to investigate the mechanisms underlying the observed neural changes.

Quinolinic acid injection in mouse medial prefrontal cortex affects reversal learning abilities, cortical connectivity and hippocampal synaptic plasticity.

Intracerebral injection of the excitotoxic, endogenous tryptophan metabolite, quinolinic acid (QA), constitutes a chemical model of neurodegenerative brain disease. Complementary techniques were combined to examine the consequences of QA injection into medial prefrontal cortex (mPFC) of C57BL6 mice. In accordance with the NMDAR-mediated synapto- and neurotoxic action of QA, we found an initial increase in excitability and an augmentation of hippocampal long-term potentiation, converting within two weeks into a reduction and impairment, respectively, of these processes. QA-induced mPFC excitotoxicity impaired behavioral flexibility in a reversal variant of the hidden-platform Morris water maze (MWM), whereas regular, extended MWM training was unaffected. QA-induced mPFC damage specifically affected the spatial-cognitive strategies that mice use to locate the platform during reversal learning. These behavioral and cognitive defects coincided with changes in cortical functional connectivity (FC) and hippocampal neuroplasticity. FC between various cortical regions was assessed by resting-state fMRI (rsfMRI) methodology, and mice that had received QA injection into mPFC showed increased FC between various cortical regions. mPFC and hippocampus (HC) are anatomically as well as functionally linked as part of a cortical network that controls higher-order cognitive functions. Together, these observations demonstrate the central functional importance of rodent mPFC as well as the validity of QA-induced mPFC damage as a preclinical rodent model of the early stages of neurodegeneration.

Functional magnetic resonance imaging (FMRI) with auditory stimulation in songbirds.

The neurobiology of birdsong, as a model for human speech, is a pronounced area of research in behavioral neuroscience. Whereas electrophysiology and molecular approaches allow the investigation of either different stimuli on few neurons, or one stimulus in large parts of the brain, blood oxygenation level dependent (BOLD) functional Magnetic Resonance Imaging (fMRI) allows combining both advantages, i.e. compare the neural activation induced by different stimuli in the entire brain at once. fMRI in songbirds is challenging because of the small size of their brains and because their bones and especially their skull comprise numerous air cavities, inducing important susceptibility artifacts. Gradient-echo (GE) BOLD fMRI has been successfully applied to songbirds (1-5) (for a review, see (6)). These studies focused on the primary and secondary auditory brain areas, which are regions free of susceptibility artifacts. However, because processes of interest may occur beyond these regions, whole brain BOLD fMRI is required using an MRI sequence less susceptible to these artifacts. This can be achieved by using spin-echo (SE) BOLD fMRI (7,8) . In this article, we describe how to use this technique in zebra finches (Taeniopygia guttata), which are small songbirds with a bodyweight of 15-25 g extensively studied in behavioral neurosciences of birdsong. The main topic of fMRI studies on songbirds is song perception and song learning. The auditory nature of the stimuli combined with the weak BOLD sensitivity of SE (compared to GE) based fMRI sequences makes the implementation of this technique very challenging.

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.

Current state-of-the-art of auditory functional MRI (fMRI) on zebra finches: technique and scientific achievements.

Songbirds provide an excellent model system exhibiting vocal learning associated with an extreme brain plasticity linked to quantifiable behavioral changes. This animal model has thus far been intensively studied using electrophysiological, histological and molecular mapping techniques. However, these approaches do not provide a global view of the brain and/or do not allow repeated measures, which are necessary to establish correlations between alterations in neural substrate and behavior. In contrast, functional Magnetic Resonance Imaging (fMRI) is a non-invasive in vivo technique which allows one (i) to study brain function in the same subject over time, and (ii) to address the entire brain at once. During the last decades, fMRI has become one of the most popular neuroimaging techniques in cognitive neuroscience for the study of brain activity during various tasks ranging from simple sensory-motor to highly cognitive tasks. By alternating various stimulation periods with resting periods during scanning, resting and task-specific regional brain activity can be determined with this technique. Despite its obvious benefits, fMRI has, until now, only been sparsely used to study cognition in non-human species such as songbirds. The Bio-Imaging Lab (University of Antwerp, Belgium) was the first to implement Blood Oxygen Level Dependent (BOLD) fMRI in songbirds - and in particular zebra finches - for the visualization of sound perception and processing in auditory and song control brain regions. The present article provides an overview of the establishment and optimization of this technique in our laboratory and of the resulting scientific findings. The introduction of fMRI in songbirds has opened new research avenues that permit experimental analysis of complex sensorimotor and cognitive processes underlying vocal communication in this animal model.

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.

Functional MRI of auditory responses in the zebra finch forebrain reveals a hierarchical organisation based on signal strength but not selectivity.

BACKGROUND: Male songbirds learn their songs from an adult tutor when they are young. A network of brain nuclei known as the 'song system' is the likely neural substrate for sensorimotor learning and production of song, but the neural networks involved in processing the auditory feedback signals necessary for song learning and maintenance remain unknown. Determining which regions show preferential responsiveness to the bird's own song (BOS) is of great importance because neurons sensitive to self-generated vocalisations could mediate this auditory feedback process. Neurons in the song nuclei and in a secondary auditory area, the caudal medial mesopallium (CMM), show selective responses to the BOS. The aim of the present study is to investigate the emergence of BOS selectivity within the network of primary auditory sub-regions in the avian pallium. METHODS AND FINDINGS: Using blood oxygen level-dependent (BOLD) fMRI, we investigated neural responsiveness to natural and manipulated self-generated vocalisations and compared the selectivity for BOS and conspecific song in different sub-regions of the thalamo-recipient area Field L. Zebra finch males were exposed to conspecific song, BOS and to synthetic variations on BOS that differed in spectro-temporal and/or modulation phase structure. We found significant differences in the strength of BOLD responses between regions L2a, L2b and CMM, but no inter-stimuli differences within regions. In particular, we have shown that the overall signal strength to song and synthetic variations thereof was different within two sub-regions of Field L2: zone L2a was significantly more activated compared to the adjacent sub-region L2b. CONCLUSIONS: Based on our results we suggest that unlike nuclei in the song system, sub-regions in the primary auditory pallium do not show selectivity for the BOS, but appear to show different levels of activity with exposure to any sound according to their place in the auditory processing stream.

Functional magnetic resonance imaging in zebra finch discerns the neural substrate involved in segregation of conspecific song from background noise.

Recently, fMRI was introduced in a well-documented animal model for vocal learning, the songbird. Using fMRI and conspecific signals mixed with different levels of broadband noise, we now demonstrate auditory-induced activation representing discriminatory properties of auditory forebrain regions in anesthetized male zebra finches (Taeniopygia guttata). Earlier behavioral tests showed comparable calling responses to the original conspecific song stimulus heard outside and inside the magnet. A significant fMRI response was elicited by conspecific song in the primary auditory thalamo-recipient subfield L2a; in neighboring subareas L2b, L3, and L; and in the rostral part of the higher-order auditory area NCM (caudomedial nidopallium). Temporal BOLD response clustering revealed rostral and caudal clusters that we defined as "cluster Field L" and "cluster NCM", respectively. However, because the actual border between caudal Field L subregions and NCM cannot be seen in the structural MR image and is not precisely reported elsewhere, the cluster NCM might also contain subregion L and the medial extremes of the subregions L2b and L3. Our results show that whereas in cluster Field L the response was not reduced by added noise, in cluster NCM the response was reduced and finally disappeared with increasing levels of noise added to the song stimulus. The activation in cluster NCM was significant for only two experimental stimuli that showed significantly more behavioral responses than the more degraded stimuli, suggesting that the first area within the auditory system where the ability to discern song from masking noise emerges is located in cluster NCM.

Neural representation of spectral and temporal features of song in the auditory forebrain of zebra finches as revealed by functional MRI.

Song perception in songbirds, just as music and speech perception in humans, requires processing the spectral and temporal structure found in the succession of song-syllables. Using functional magnetic resonance imaging and synthetic songs that preserved exclusively either the temporal or the spectral structure of natural song, we investigated how vocalizations are processed in the avian forebrain. We found bilateral and equal activation of the primary auditory region, field L. The more ventral regions of field L showed depressed responses to the synthetic songs that lacked spectral structure. These ventral regions included subarea L3, medial-ventral subarea L and potentially the secondary auditory region caudal medial nidopallium. In addition, field L as a whole showed unexpected increased responses to the temporally filtered songs and this increase was the largest in the dorsal regions. These dorsal regions included L1 and the dorsal subareas L and L2b. Therefore, the ventral region of field L appears to be more sensitive to the preservation of both spectral and temporal information in the context of song processing. We did not find any differences in responses to playback of the bird's own song vs other familiar conspecific songs. We also investigated the effect of three commonly used anaesthetics on the blood oxygen level-dependent response: medetomidine, urethane and isoflurane. The extent of the area activated and the stimulus selectivity depended on the type of anaesthetic. We discuss these results in the context of what is known about the locus of action of the anaesthetics, and reports of neural activity measured in electrophysiological experiments.

Current status of functional MRI on small animals: application to physiology, pathophysiology, and cognition.

This review aims to make the reader aware of the potential of functional MRI (fMRI) in brain activation studies in small animal models. As small animals generally require anaesthesia for immobilization during MRI protocols, this is believed to be a serious limitation to the type of question that can be addressed with fMRI. We intend to introduce a fresh view with an in-depth overview of the surprising number of fMRI applications in a wide range of important research domains in neuroscience. These include the pathophysiology of brain functioning, the basic science of activity, and functional connectivity of different sensory circuits, including sensory brain mapping, the challenges when studying the hypothalamus as the major control centre in the central nervous system, and the limbic system as neural substrate for emotions and reward. Finally the contribution of small animal fMRI research to cognitive neuroscience is outlined. This review avoids focusing exclusively on traditional small laboratory animals such as rodents, but rather aims to broaden the scope by introducing alternative lissencephalic animal models such as songbirds and fish, as these are not yet well recognized as neuroimaging study subjects. These models are well established in many other neuroscience disciplines, and this review will show that their investigation with in vivo imaging tools will open new doors to cognitive neuroscience and the study of the autonomous nervous system in experimental animals.

Spatiotemporal properties of the BOLD response in the songbirds' auditory circuit during a variety of listening tasks.

Auditory fMRI in humans has recently received increasing attention from cognitive neuroscientists as a tool to understand mental processing of learned acoustic sequences and analyzing speech recognition and development of musical skills. The present study introduces this tool in a well-documented animal model for vocal learning, the songbird, and provides fundamental insight in the main technical issues associated with auditory fMRI in these songbirds. Stimulation protocols with various listening tasks lead to appropriate activation of successive relays in the songbirds' auditory pathway. The elicited BOLD response is also region and stimulus specific, and its temporal aspects provide accurate measures of the changes in brain physiology induced by the acoustic stimuli. Extensive repetition of an identical stimulus does not lead to habituation of the response in the primary or secondary telencephalic auditory regions of anesthetized subjects. The BOLD signal intensity changes during a stimulation and subsequent rest period have a very specific time course which shows a remarkable resemblance to auditory evoked BOLD responses commonly observed in human subjects. This observation indicates that auditory fMRI in the songbird may establish a link between auditory related neuro-imaging studies done in humans and the large body of neuro-ethological research on song learning and neuro-plasticity performed in songbirds.