Beyond the surface: how ex-vivo diffusion-weighted imaging reveals large animal brain microstructure and connectivity.

Diffusion-weighted Imaging (DWI) is an effective and state-of-the-art neuroimaging method that non-invasively reveals the microstructure and connectivity of tissues. Recently, novel applications of the DWI technique in studying large brains through ex-vivo imaging enabled researchers to gain insights into the complex neural architecture in different species such as those of Perissodactyla (e.g., horses and rhinos), Artiodactyla (e.g., bovids, swines, and cetaceans), and Carnivora (e.g., felids, canids, and pinnipeds). Classical in-vivo tract-tracing methods are usually considered unsuitable for ethical and practical reasons, in large animals or protected species. Ex-vivo DWI-based tractography offers the chance to examine the microstructure and connectivity of formalin-fixed tissues with scan times and precision that is not feasible in-vivo. This paper explores DWI's application to ex-vivo brains of large animals, highlighting the unique insights it offers into the structure of sometimes phylogenetically different neural networks, the connectivity of white matter tracts, and comparative evolutionary adaptations. Here, we also summarize the challenges, concerns, and perspectives of ex-vivo DWI that will shape the future of the field in large brains.

Functional MRI of imprinting memory: a new avenue for neurobiology of early learning.

Filial imprinting, a crucial ethological paradigm, provides insights into the neurobiology of early learning and its long-term impact on behaviour. To date, only invasive techniques, such as autoradiography or lesion, have been employed to understand this behaviour. The primary limitation of these methods lies in their constrained access to the entire brain, impeding the exploration of brain networks crucial at various stages of this paradigm. Recently, advances in functional magnetic resonance imaging (fMRI) in the avian brain have opened new windows to explore bird's brain function at the network level. Here, we developed a ground-breaking non-invasive functional MRI technique for awake, newly hatched chicks that record whole-brain BOLD signal changes throughout imprinting experiments. While the initial phases of memory acquisition imprinting behaviour have been unravelled, the long-term storage and retrieval components of imprinting memories are still unknown. Our findings identified potential long-term storage of imprinting memories across a neural network, including the hippocampal formation, the medial striatum, the arcopallium, and the prefrontal-like nidopallium caudolaterale. This platform opens up new avenues for exploring the broader landscape of learning and memory processes in neonatal vertebrates, contributing to a more comprehensive understanding of the intricate interplay between behaviour and brain networks.

Constrained spherical deconvolution on diffusion-weighted images of dolphin brains.

Invasive neuronal tract-tracing is not permitted in very large or endangered animals. This is especially the case in marine mammals like dolphins. Diffusion-weighted imaging of fiber tracts could be an alternative if feasible even in brains that have been fixed in formalin for a long time. This currently is a problem, especially for detecting crossing fibers. We applied a state-of-the-art algorithm of Diffusion-weighted imaging called Constrained Spherical Deconvolution on diffusion data of three fixed brains of bottlenose dolphins using clinical human MRI parameters and were able to identify complex fiber patterns within a voxel. Our findings indicate that in order to maintain the structural integrity of the tissue, short-term post-mortem fixation is necessary. Furthermore, pre-processing steps are essential to remove the classical Diffusion-weighted imaging artifacts from images: however, the algorithm is still able to resolve fiber tracking in regions with various signal intensities. The described imaging technique reveals complex fiber patterns in cetacean brains that have been preserved in formalin for extended periods of time and thus opens a new window into our understanding of cetacean neuroanatomy.

Segmented spin-echo echo-planar imaging improves whole-brain BOLD functional MRI in awake pigeon brains.

Functional magnetic resonance imaging (fMRI) in awake small animals such as pigeons or songbirds opens a new window into the neural fundaments of cognitive behavior. However, high-field fMRI in the avian brain is challenging due to strong local magnetic field inhomogeneities caused by air cavities in the skull. A spoiled gradient-echo fMRI sequence has already been used to map the auditory network in songbirds, but due to susceptibility artifacts only 50% of the whole brain could be recorded. Since whole-brain fMRI coverage is vital to reveal whole-brain networks, an MRI sequence that is less susceptible to these artifacts was required. This was recently achieved in various bird species by using a rapid acquisition with relaxation enhancement (RARE) sequence. Weak blood oxygen level-dependent (BOLD) sensitivity, low temporal resolution, and heat caused by the long train of RF refocusing pulses are the main limits of RARE fMRI at high magnetic fields. To go beyond some of these limitations, we here describe the implementation of a two-segmented spin-echo echo-planar imaging (SE-EPI). The proposed sequence covers the whole brain of awake pigeons. The sequence was applied to investigate the auditory network in awake pigeons and assessed the relative merits of this method in comparison with the single-shot RARE sequence. At the same imaging resolution but with a volume acquisition of 3 s versus 4 s for RARE, the two-segmented SE-EPI provided twice the strength of BOLD activity compared with the single-shot RARE sequence, while the image signal-to-noise ratio (SNR) and in particular the temporal SNR were very similar for the two sequences. In addition, the activation patterns in two-segmented SE-EPI data are more symmetric and larger than single-shot RARE results. Two-segmented SE-EPI represents a valid alternative to the RARE sequence in avian fMRI research since it yields more than twice the BOLD sensitivity per unit of time with much less energy deposition and better temporal resolution, particularly for event-related experiments.

The prefrontal cortex of the bottlenose dolphin (Tursiops truncatus Montagu, 1821): a tractography study and comparison with the human.

Cetaceans are well known for their remarkable cognitive abilities including self-recognition, sound imitation and decision making. In other mammals, the prefrontal cortex (PFC) takes a key role in such cognitive feats. In cetaceans, however, a PFC could up to now not be discerned based on its usual topography. Classical in vivo methods like tract tracing are legally not possible to perform in Cetacea, leaving diffusion-weighted imaging (DWI) as the most viable alternative. This is the first investigation focussed on the identification of the cetacean PFC homologue. In our study, we applied the constrained spherical deconvolution (CSD) algorithm on 3 T DWI scans of three formalin-fixed brains of bottlenose dolphins (Tursiops truncatus) and compared the obtained results to human brains, using the same methodology. We first identified fibres related to the medio-dorsal thalamic nuclei (MD) and then seeded the obtained putative PFC in the dolphin as well as the known PFC in humans. Our results outlined the dolphin PFC in areas not previously studied, in the cranio-lateral, ectolateral and opercular gyri, and furthermore demonstrated a similar connectivity pattern between the human and dolphin PFC. The antero-lateral rotation of the PFC, like in other areas, might be the result of the telescoping process which occurred in these animals during evolution.

Wide-spread brain activation and reduced CSF flow during avian REM sleep.

Mammalian sleep has been implicated in maintaining a healthy extracellular environment in the brain. During wakefulness, neuronal activity leads to the accumulation of toxic proteins, which the glymphatic system is thought to clear by flushing cerebral spinal fluid (CSF) through the brain. In mice, this process occurs during non-rapid eye movement (NREM) sleep. In humans, ventricular CSF flow has also been shown to increase during NREM sleep, as visualized using functional magnetic resonance imaging (fMRI). The link between sleep and CSF flow has not been studied in birds before. Using fMRI of naturally sleeping pigeons, we show that REM sleep, a paradoxical state with wake-like brain activity, is accompanied by the activation of brain regions involved in processing visual information, including optic flow during flight. We further demonstrate that ventricular CSF flow increases during NREM sleep, relative to wakefulness, but drops sharply during REM sleep. Consequently, functions linked to brain activation during REM sleep might come at the expense of waste clearance during NREM sleep.

Unihemispheric evidence accumulation in pigeons.

Perceptual decision making involves choices between alternatives based on sensory information. Studies in primates and rodents revealed a stochastic perceptual evidence accumulation process that, after reaching threshold, results in action execution. Birds represent a cognitively highly successful vertebrate class that has been evolving independent from mammals for more than 300 million years. The present study investigated whether perceptual decision making in pigeons shows behavioral and computational dynamics comparable to those in mammals and rodents. Using a novel "pigeon helmet" with liquid shutter displays that controls visual input to individual eyes/hemispheres with precise timing, we indeed revealed highly similar dynamics of perceptual decision making. Thus, both mammals and birds seem to share this core cognitive process that possibly represents a fundamental constituent of decision making throughout vertebrates. Interestingly, in our experiments we additionally discovered that both avian hemispheres start independent sensory accumulation processes without any major interhemispheric exchange. Because birds lack a corpus callosum and have only a small anterior commissure, they seem to be forced to decide on motor responses based on unihemispheric decisions under conditions of time pressure. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Visual and Tactile Sensory Systems Share Common Features in Object Recognition.

Although we use our visual and tactile sensory systems interchangeably for object recognition on a daily basis, little is known about the mechanism underlying this ability. This study examined how 3D shape features of objects form two congruent and interchangeable visual and tactile perceptual spaces in healthy male and female participants. Since active exploration plays an important role in shape processing, a virtual reality environment was used to visually explore 3D objects called digital embryos without using the tactile sense. In addition, during the tactile procedure, blindfolded participants actively palpated a 3D-printed version of the same objects with both hands. We first demonstrated that the visual and tactile perceptual spaces were highly similar. We then extracted a series of 3D shape features to investigate how visual and tactile exploration can lead to the correct identification of the relationships between objects. The results indicate that both modalities share the same shape features to form highly similar veridical spaces. This finding suggests that visual and tactile systems might apply similar cognitive processes to sensory inputs that enable humans to rely merely on one modality in the absence of another to recognize surrounding objects.

Gradual Restraint Habituation for Awake Functional Magnetic Resonance Imaging Combined With a Sparse Imaging Paradigm Reduces Motion Artifacts and Stress Levels in Rodents.

Functional magnetic resonance imaging, as a non-invasive technique, offers unique opportunities to assess brain function and connectivity under a broad range of applications, ranging from passive sensory stimulation to high-level cognitive abilities, in awake animals. This approach is confounded, however, by the fact that physical restraint and loud unpredictable acoustic noise must inevitably accompany fMRI recordings. These factors induce marked stress in rodents, and stress-related elevations of corticosterone levels are known to alter information processing and cognition in the rodent. Here, we propose a habituation strategy that spans specific stages of adaptation to restraint, MRI noise, and confinement stress in awake rats and circumvents the need for surgical head restraint. This habituation protocol results in stress levels during awake fMRI that do not differ from pre-handling levels and enables stable image acquisition with very low motion artifacts. For this, rats were gradually trained over a period of three weeks and eighteen training sessions. Stress levels were assessed by analysis of fecal corticosterone metabolite levels and breathing rates. We observed significant drops in stress levels to below pre-handling levels at the end of the habituation procedure. During fMRI in awake rats, after the conclusion of habituation and using a non-invasive head-fixation device, breathing was stable and head motion artifacts were minimal. A task-based fMRI experiment, using acoustic stimulation, conducted 2 days after the end of habituation, resulted in precise whole brain mapping of BOLD signals in the brain, with clear delineation of the expected auditory-related structures. The active discrimination by the animals of the acoustic stimuli from the backdrop of scanner noise was corroborated by significant increases in BOLD signals in the thalamus and reticular formation. Taken together, these data show that effective habituation to awake fMRI can be achieved by gradual and incremental acclimatization to the experimental conditions. Subsequent BOLD recordings, even during superimposed acoustic stimulation, reflect low stress-levels, low motion and a corresponding high-quality image acquisition. Furthermore, BOLD signals obtained during fMRI indicate that effective habituation facilitates selective attention to sensory stimuli that can in turn support the discrimination of cognitive processes in the absence of stress confounds.

Event-related functional MRI of awake behaving pigeons at 7T.

Animal-fMRI is a powerful method to understand neural mechanisms of cognition, but it remains a major challenge to scan actively participating small animals under low-stress conditions. Here, we present an event-related functional MRI platform in awake pigeons using single-shot RARE fMRI to investigate the neural fundaments for visually-guided decision making. We established a head-fixated Go/NoGo paradigm, which the animals quickly learned under low-stress conditions. The animals were motivated by water reward and behavior was assessed by logging mandibulations during the fMRI experiment with close to zero motion artifacts over hundreds of repeats. To achieve optimal results, we characterized the species-specific hemodynamic response function. As a proof-of-principle, we run a color discrimination task and discovered differential neural networks for Go-, NoGo-, and response execution-phases. Our findings open the door to visualize the neural fundaments of perceptual and cognitive functions in birds-a vertebrate class of which some clades are cognitively on par with primates.

A three-dimensional digital atlas of the Nile crocodile (Crocodylus niloticus) forebrain.

The phylogenetic position of crocodilians in relation to birds and mammals makes them an interesting animal model for investigating the evolution of the nervous system in amniote vertebrates. A few neuroanatomical atlases are available for reptiles, but with a growing interest in these animals within the comparative neurosciences, a need for these anatomical reference templates is becoming apparent. With the advent of MRI being used more frequently in comparative neuroscience, the aim of this study was to create a three-dimensional MRI-based atlas of the Nile crocodile (Crocodylus niloticus) brain to provide a common reference template for the interpretation of the crocodilian, and more broadly reptilian, brain. Ex vivo MRI acquisitions in combination with histological data were used to delineate crocodilian brain areas at telencephalic, diencephalic, mesencephalic, and rhombencephalic levels. A total of 50 anatomical structures were successfully identified and outlined to create a 3-D model of the Nile crocodile brain. The majority of structures were more readily discerned within the forebrain of the crocodile with the methods used to produce this atlas. The anatomy outlined herein corresponds with both classical and recent crocodilian anatomical analyses, barring a few areas of contention predominantly related to a lack of functional data and conflicting nomenclature.

Functional MRI in the Nile crocodile: a new avenue for evolutionary neurobiology.

Crocodilians are important for understanding the evolutionary history of amniote neural systems as they are the nearest extant relatives of modern birds and share a stem amniote ancestor with mammals. Although the crocodilian brain has been investigated anatomically, functional studies are rare. Here, we employed functional magnetic resonance imaging (fMRI), never tested in poikilotherms, to investigate crocodilian telencephalic sensory processing. Juvenile Crocodylus niloticus were placed in a 7 T MRI scanner to record blood oxygenation level-dependent (BOLD) signal changes during the presentation of visual and auditory stimuli. Visual stimulation increased BOLD signals in rostral to mid-caudal portions of the dorso-lateral anterior dorsal ventricular ridge (ADVR). Simple auditory stimuli led to signal increase in the rostromedial and caudocentral ADVR. These activation patterns are in line with previously described projection fields of diencephalic sensory fibres. Furthermore, complex auditory stimuli activated additional regions of the caudomedial ADVR. The recruitment of these additional, presumably higher-order, sensory areas reflects observations made in birds and mammals. Our results indicate that structural and functional aspects of sensory processing have been likely conserved during the evolution of sauropsids. In addition, our study shows that fMRI can be used to investigate neural processing in poikilotherms, providing a new avenue for neurobiological research in these critical species.

Decoding the different states of visual attention using functional and effective connectivity features in fMRI data.

The present paper concentrates on the impact of visual attention task on structure of the brain functional and effective connectivity networks using coherence and Granger causality methods. Since most studies used correlation method and resting-state functional connectivity, the task-based approach was selected for this experiment to boost our knowledge of spatial and feature-based attention. In the present study, the whole brain was divided into 82 sub-regions based on Brodmann areas. The coherence and Granger causality were applied to construct functional and effective connectivity matrices. These matrices were converted into graphs using a threshold, and the graph theory measures were calculated from it including degree and characteristic path length. Visual attention was found to reveal more information during the spatial-based task. The degree was higher while performing a spatial-based task, whereas characteristic path length was lower in the spatial-based task in both functional and effective connectivity. Primary and secondary visual cortex (17 and 18 Brodmann areas) were highly connected to parietal and prefrontal cortex while doing visual attention task. Whole brain connectivity was also calculated in both functional and effective connectivity. Our results reveal that Brodmann areas of 17, 18, 19, 46, 3 and 4 had a significant role proving that somatosensory, parietal and prefrontal regions along with visual cortex were highly connected to other parts of the cortex during the visual attention task. Characteristic path length results indicated an increase in functional connectivity and more functional integration in spatial-based attention compared with feature-based attention. The results of this work can provide useful information about the mechanism of visual attention at the network level.

In vivo measurement of T1 and T2 relaxation times in awake pigeon and rat brains at 7T.

PURPOSE: Establishment of regional longitudinal (T1 ) and transverse (T2 ) relaxation times in awake pigeons and rats at 7T field strength. Regional differences in relaxation times between species and between two different pigeon breeds (homing pigeons and Figurita pigeons) were investigated. METHODS: T1 and T2 relaxation times were determined for nine functionally equivalent brain regions in awake pigeons and rats using a multiple spin-echo saturation recovery method with variable repetition time and a multi-slice/multi-echo sequence, respectively. Optimized head fixation and habituation protocols were applied to accustom animals to the scanning conditions and to minimize movement. RESULTS: The habituation protocol successfully limited movement of the awake animals to a negligible minimum, allowing reliable measurement of T1 and T2 values within all regions of interest. Significant differences in relaxation times were found between rats and pigeons but not between different pigeon breeds. CONCLUSION: The obtained T1 and T2 values for awake pigeons and rats and the optimized habituation protocol will augment future MRI studies with awake animals. The differences in relaxation times observed between species underline the importance of the acquisition of T1 /T2 values as reference points for specific experiments. Magn Reson Med 79:1090-1100, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Effect of airway remodeling and hyperresponsiveness on complexity of breathing pattern in rat.

The complexity of respiratory dynamics is decreased, in association with disease severity, in patients with asthma. However, the pathophysiological basis of decreased complexity of breathing pattern in asthma is not clear. In the present study, we investigated the effect of airway remodeling and hyperresponsiveness induced by repeated bronchoconstriction (using methacholine) on breathing pattern in rats with or without allergen-induced sensitization. Entropy analysis of respiratory variability showed decreased irregularity (less complexity) of respiratory rhythm in this rat model of asthma. Airway remodeling and hyperresponsiveness induced by repeated bronchoconstriction also led to increased regularity of respiratory dynamics in sensitized rats. However, these airway alterations had no significant effect on the complexity of breathing pattern in non-sensitized rats. Our results indicate that mechanical respiratory alterations cannot per se attenuate the complexity of respiratory dynamics, unless there is an underlying inflammation. We suggest further studies on underlying mechanisms of breathing variability with focus on respiratory control alterations due to airway inflammation.

Functional Connectivity Pattern of the Internal Hippocampal Network in Awake Pigeons: A Resting-State fMRI Study.

In the last two decades, the avian hippocampus has been repeatedly studied with respect to its architecture, neurochemistry, and connectivity pattern. We review these insights and conclude that we unfortunately still lack proper knowledge on the interaction between the different hippocampal subregions. To fill this gap, we need information on the functional connectivity pattern of the hippocampal network. These data could complement our structural connectivity knowledge. To this end, we conducted a resting-state fMRI experiment in awake pigeons in a 7-T MR scanner. A voxel-wise regression analysis of blood oxygenation level-dependent (BOLD) fluctuations was performed in 6 distinct areas, dorsomedial (DM), dorsolateral (DL), triangular shaped (Tr), dorsolateral corticoid (CDL), temporo-parieto-occipital (TPO), and lateral septum regions (SL), to establish a functional connectivity map of the avian hippocampal network. Our study reveals that the system of connectivities between CDL, DL, DM, and Tr is the functional backbone of the pigeon hippocampal system. Within this network, DM is the central hub and is strongly associated with DL and CDL BOLD signal fluctuations. DM is also the only hippocampal region to which large Tr areas are functionally connected. In contrast to published tracing data, TPO and SL are only weakly integrated in this network. In summary, our findings uncovered a structurally otherwise invisible architecture of the avian hippocampal formation by revealing the dynamic blueprints of this network.

Bronchoconstriction Induces Structural and Functional Airway Alterations in Non-sensitized Rats.

The impact of mechanical forces on pathogenesis of airway remodeling and the functional consequences in asthma remains to be fully established. In the present study, we investigated the effect of repeated bronchoconstriction induced by methacholine (MCh) on airway remodeling and airway hyperresponsiveness (AHR) in rats with or without sensitization to an external allergen. We provide evidence that repeated bronchoconstriction, using MCh, alone induces airway inflammation and remodeling as well as AHR in non-allergen-sensitized rats. Also, we found that the airways are structurally and functionally altered by bronchoconstriction induced by either allergen or MCh in allergen-sensitized animals. This finding provides a new animal model for the development of airway remodeling and AHR in mammals and can be used for studying the complex reciprocal relationship between bronchoconstriction and airway inflammation. Further studies on presented animal models are required to clarify the exact mechanisms underlying airway remodeling due to bronchoconstriction and the functional consequences.

EEG phase patterns reflect the representation of semantic categories of objects.

Oscillations of electroencephalographic signals represent the cognitive processes arose from the behavioral task and sensory representations across the mental state activity. Previous studies have shown the relation between event-related EEG and sensory-cognitive representation and revealed that categorization of presented object can be successfully recognized using recorded EEG signals when subjects view objects. Here, EEG signals in conjunction with a multivariate pattern recognition technique were used for investigating the possibility to identify conceptual representation based on the presentation of 12 semantic categories of objects (5 exemplars per category). Using multivariate stimulus decoding methods, surprisingly, we demonstrate that how objects are discriminated from phase pattern of EEG signals across the time in low-frequency band (1-4 Hz), but not from power of oscillatory brain signals in the same frequency band. In contrast, discrimination accuracy from the power of EEG signals has significantly higher than the performance from phase of EEG signal in the high-frequency band (20-30 Hz). Moreover, our results indicate that how the accuracy of prediction changes between various areas of brain continuously across the time. In particular, we find that, during the object categorization task, the inter-trial phase coherence in low-frequency band is significantly higher than other frequency in various regions of interests. This measure is associated with decoding pattern across the time. These results suggest that the mechanism underlying conceptual representation can be mediated by the phase of oscillatory neural activity.

RDLPFC area of the brain encodes sentence polarity: a study using fMRI.

In this study, we use functional magnetic resonance imaging (fMRI) in combination with multivoxel pattern analysis to address the question of how mental activities that correspond to sentence polarity (affirmative or negative sentences) are encoded in the brain. This approach allows us to investigate the role of left/right dorsolateral prefrontal cortex (DLPFC) in predicting the neural activity of fMRI associated with sentence polarities. Subjects in the experiment were asked to judge the matching of the presented picture with the meaning of affirmative and negative sentences. Our results highlight the role of RDLPFC in encoding of the related mental activity to sentence polarities such that the right hemisphere (RDLPFC) can predict sentence polarity with high accuracy as compared to the left hemisphere (LDLPFC), and that the negative sentences are decoded with high performance as compared to affirmative sentences from the RDLPFC across subjects. In addition, this experiment's results show that negative sentences involve more syntactic structure than affirmative sentences.