Significance:Adapting optical imaging technology to avian models can overcome many limitations imposed by functional magnetic resonance imaging (fMRI), which currently restricts the number of species used to study functional connectivity. Developing advanced technology to expand the diversity of species that can be effectively imaged is crucial for addressing significant questions that are currently unreachable, such as understanding the evolution of cognition from a comparative perspective. Aim:We assessed the potential of optical imaging technology to measure functional connectivity in birds, utilizing pigeons as an avian model. We evaluated whether we could partition the dorsal surface of the pigeon brain into units that correspond to known anatomical regions. Finally, we compared our results with those obtained from a separate dataset acquired using fMRI. Approach:Using optical intrinsic signal imaging, a widefield optical imaging method, we imaged resting state functional connectivity in scalp-retracted anesthetized pigeons. We then used iterative parcellation and hierarchical clustering to create functional connectivity maps of correlation between parcels at two spatial scales. We recorded a second independent dataset of ten pigeons using a single-shot multi-slice gradient echo EPI sequence fMRI and applied the same parcellation method to compare functional connectivity patterns between the two methodologies. Results:We successfully partitioned signal activity into clusters of parcels that exhibit left-right symmetry between hemispheres and which align well with known anatomical regions of the dorsal surface of the pigeon brain. Moreover, functional connectivity matrices reveal positive correlations between homotopic regions. These cluster partitions and functional connectivity maps display similar patterns across and within individuals. Finally, WOI imaging results were comparable to resting state data acquired using fMRI. Conclusions:Taken together, these results demonstrate the potential of optical imaging technology for the reliable and cost-effective characterization of functional connectivity in birds. In addition, they position optical imaging methods as a valuable tool for large-scale comparative and network-level studies in this taxon.

Extinction learning is an important learning process that enables adaptive and flexible behavior. Human neuroimaging studies show that the neural basis of extinction learning consists of a neural network that includes the hippocampus, amygdala, and subcomponents of the prefrontal cortex, but also extends beyond them. The limitations of applying fMRI to actively participating animals have so far restricted the identification of the entire extinction network in non-human animals. Here, we present the first fMRI study of extinction in awake and actively participating pigeons, using a Go/NoGo operant paradigm with a water reward. Our study revealed an extensive and largely left hemispheric telencephalic network of sensory, limbic, executive, and motor areas that slowly ceased to be active during the process of extinction learning. We propose that the beginning of extinction ignites a neuronal updating of the associated consequences of own actions within a large telencephalic neural network until a new association is established which competes with the previously acquired operant response.

Abstract Ex vivo diffusion weighted imaging (DWI) enables high-resolution characterization of brain connectivity and is increasingly applied in comparative and evolutionary neuroscience. However, variability in tissue preparation and contrast agent exposure can substantially affect relaxation properties and compromise reproducibility, particularly in non-mammalian species. Here, we systematically assess the impact of different gadolinium-based contrast agent exposure protocols on relaxation stability and DWI compatibility in fixed pigeon brains. Brains were perfusion-fixed with 2% paraformaldehyde and assigned to four preparation protocols: (i) contrast agent exposure during perfusion, post-fixation, and rehydration; (ii) post-fixation and rehydration only; (iii) rehydration only; (iv) no contrast agent. Quantitative T1, T2, T2*, and DWI data were acquired at five time points over 70 days using a 7T MRI system. Protocols involving contrast agent during perfusion or post-fixation produced comparable relaxation trajectories, with T1, T2, and T2* stabilizing by Day 13. On day 13 the T1 values of tissue that was exposed to contrast agent, regardless of the application protocol were between 230.86 ms and 266.89 ms, while the T1 values of the control group were over 1100 ms at this point in time. T2 values of the experimental groups were between 39.97 ms and 56.17 ms while T2 values of the control group were between 58.68 ms and 77.82 ms. T2* values of the experimental groups were between 27.27 ms and 43.33 ms while T2* values of the control group were between 46.16 ms and 65.93 ms. Importantly, contrast agent exposure during rehydration alone resulted in equivalent stabilization after two weeks, reflecting gradual contrast agent diffusion into the tissue. In contrast, control samples without contrast agent exhibited significantly elevated T2 and T2* at later time points. These results demonstrate that post-fixation contrast agent exposure during rehydration is sufficient to achieve stable relaxation parameters and DWI compatibility, assessed via fractional anisotropy (FA) and mean diffusivity (MD) in ex vivo avian brain tissue. This minimal preparation protocol enhances reproducibility, reduces handling complexity, and supports standardized cross-species neuroimaging of brain connectivity.

Calpainopathy is a rare genetic myopathy without causal treatment available. Recent advances have produced promising treatment strategies, including genetic treatment and immunomodulation, that are currently being tested pre-clinically in murine models. Traditional behavioral assays frequently fail to detect early motor deficits in corresponding mouse models. Thus, this study investigated whether more sophisticated measures, such as quantitative magnetic resonance imaging (qMRI) and automated motor assessments can reveal early pathological changes in a mouse model of calpainopathy, particularly in the subclinical stages. In this study, Calpain 3-deficient mice (Capn3-deficient) and wild-type controls underwent a standardized qMRI protocol on a 7T scanner at 5 and 15 months of age. Fat fraction (FF), water T2 relaxation time (wT2), and diffusion metrics were analyzed across selected hindlimb muscles. Additionally, voluntary motor activity was monitored using an intelligent phenotyping housing system. Motor activity assays revealed minimal and inconsistent genotype effects. Sex significantly affected qMRI measures, altering fat distribution and wT2 at 5 and 15 months. At 5 months, no significant differences were detected between genotypes. At 15 months, Capn3-deficient mice exhibited elevated wT2 in the soleus and gastrocnemius muscles, correlating with endomysial immune cell infiltration. Diffusion parameters and FF remained unchanged. Taken together, the results show that muscle qMRI, particularly T2 mapping, can sensitively detect subclinical muscle changes in aging Capn3-deficient mice, pointing towards inflammatory processes. Pronounced sex differences and measurement variability underscore the need for larger, sex-balanced cohorts. Extending follow-up beyond 15 months will likely increase sensitivity for later-stage pathology.

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.

Task-free functional connectivity in animal models provides an experimental framework to examine connectivity phenomena under controlled conditions and allows for comparisons with data modalities collected under invasive or terminal procedures. Currently, animal acquisitions are performed with varying protocols and analyses that hamper result comparison and integration. Here we introduce StandardRat, a consensus rat functional magnetic resonance imaging acquisition protocol tested across 20 centers. To develop this protocol with optimized acquisition and processing parameters, we initially aggregated 65 functional imaging datasets acquired from rats across 46 centers. We developed a reproducible pipeline for analyzing rat data acquired with diverse protocols and determined experimental and processing parameters associated with the robust detection of functional connectivity across centers. We show that the standardized protocol enhances biologically plausible functional connectivity patterns relative to previous acquisitions. The protocol and processing pipeline described here is openly shared with the neuroimaging community to promote interoperability and cooperation toward tackling the most important challenges in neuroscience. The authors pooled resources to identify best practices and develop a new standardized protocol for estimating functional connectivity in rats with magnetic resonance imaging.

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.

Quantitative muscle MRI is increasingly important in the non-invasive evaluation of neuromuscular disorders and their progression. Underlying histopathotological alterations, leading to changes in qMRI parameters are incompletely unraveled. Early microstructural differences of unknown origin reflected by Diffusion MRI in non-fat infiltrated muscles were detected in Pompe patients. This study employed a longitudinal approach with a Pompe disease mouse model to investigate the histopathological basis of these changes. Monthly scans of Pompe (Gaa 6neo/6neo ) and wildtype mice (age 1–8 months) were conducted using diffusion MRI, T2-mapping, and Dixon-based water-fat imaging on a 7 T scanner. Immunofluorescence studies on quadriceps muscles were analyzed for lysosomal accumulations and autophagic buildup and correlated with MRI outcome measures. Fat fraction and water-T2 did not differ between groups and remained stable over time. In Pompe mice, fractional anisotropy increased, while mean diffusivity (MD) and radial diffusivity (RD) decreased in all observed muscles. Autophagic marker and muscle fibre diameter revealed significant negative correlations with reduced RD and MD, while lysosomal marker did not show any change or correlation. Using qMRI, we showed diffusion changes in muscles of presymptomatic Pompe mice without fat-infiltrated muscles and correlated them to autophagic markers and fibre diameter, indicating diffusion MRI reveals autophagic buildup.

Task-free functional connectivity in animal models provides an experimental framework to examine connectivity phenomena under controlled conditions and allows comparison with invasive or terminal procedures. To date, animal acquisitions are performed with varying protocols and analyses that hamper result comparison and integration. We introduce StandardRat , a consensus rat functional MRI acquisition protocol tested across 20 centers. To develop this protocol with optimized acquisition and processing parameters, we initially aggregated 65 functional imaging datasets acquired in rats from 46 centers. We developed a reproducible pipeline for the analysis of rat data acquired with diverse protocols and determined experimental and processing parameters associated with a more robust functional connectivity detection. We show that the standardized protocol enhances biologically plausible functional connectivity patterns, relative to pre-existing acquisitions. The protocol and processing pipeline described here are openly shared with the neuroimaging community to promote interoperability and cooperation towards tackling the most important challenges in neuroscience.

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.

The Alzheimer disease-associated multifunctional low-density lipoprotein receptor-related protein-1 is expressed in the brain. Recent studies uncovered a role of this receptor for the appropriate functioning of neural stem cells, oligodendrocytes, and neurons. The constitutive knock-out (KO) of the receptor is embryonically lethal. To unravel the receptors' role in the developing brain we generated a mouse mutant by specifically targeting radial glia stem cells of the dorsal telencephalon. The low-density lipoprotein receptor-related protein-1 lineage-restricted KO female and male mice, in contrast to available models, developed a severe neurological phenotype with generalized seizures during early postnatal development. The mechanism leading to a buildup of hyperexcitability and emergence of seizures was traced to a failure in adequate astrocyte development and deteriorated postsynaptic density integrity. The detected impairments in the astrocytic lineage: precocious maturation, reactive gliosis, abolished tissue plasminogen activator uptake, and loss of functionality emphasize the importance of this glial cell type for synaptic signaling in the developing brain. Together, the obtained results highlight the relevance of astrocytic low-density lipoprotein receptor-related protein-1 for glutamatergic signaling in the context of neuron-glia interactions and stage this receptor as a contributing factor for epilepsy.

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.

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.

The number of human embryology collections is very limited worldwide. Some of these comprise the Carnegie Collection, Kyoto Collection, and the Blechschmidt Collection. One further embryonic collection is the Hinrichsen Collection of the Ruhr University Bochum, Germany, which also contains very well-preserved embryos/fetuses, along with approximately 16,000 histological sections. The digitization of this collection is indispensable to enable conservation of the collection for the future and to provide a large group of embryologists, researchers, and physicians access to these histological slides. A small selection of these scans is available at the website of the Digital Embryology Consortium [https://-human-embryology.org/wiki/Main_Page].

Background and Purpose— Detection and localization of the early phase of blood–brain barrier disruption (BBBD) in vivo during cerebral ischemia/reperfusion injury remain a major challenge but may be a relevant outcome parameter in stroke. Methods— We studied early BBBD in mice after transient middle cerebral artery occlusion by multimodal, high-field (9.4T) in vivo magnetic resonance imaging, including the contrast agent gadofluorineM as an albumin-binding tracer. GadofluorineM contrast-enhanced magnetic resonance imaging was performed to determine BBBD at 2, 6, and 24 hours after reperfusion. BBBD was confirmed and localized along the microvascular tree by using fluorescent gadofluorineM and immunofluorescence stainings (cluster of differentiation 31, ephrin type-B receptor 4, alpha smooth muscle actin, ionized calcium binding adaptor molecule 1). Results— GadofluorineM contrast-enhanced magnetic resonance imaging revealed a multifocal spatial distribution of early BBBD and its close association with the microvasculature at a resolution of 40 μm. GadofluorineM leakage was closely associated with ephrin type-B receptor 4-positive but not alpha smooth muscle actin-positive vessels. The multifocal pattern of early BBBD (already at 2 hours after reperfusion) thus occurred in the distal capillary and venular microvascular bed. These multifocal zones showed distinct imaging signs indicative of early vasogenic edema. The total volume of multifocal early BBBD accurately predicted infarct size at 24 hours after reperfusion. Conclusions— Early BBBD in focal cerebral ischemia initiates multifocally in the distal capillary and venular bed of the cerebral microvasculature. It is closely associated with perimicrovascular vasogenic edema and microglial activation and predicts the extent of final infarction.

Purpose: To investigate the association of inflammation and brain edema in a cerebral malaria (CM) mouse model with a combination of bis-5-hydroxy-tryptamide-diethylenetriaminepentaacetate gadolinium, referred to as MPO-Gd, and cross-linked iron oxide nanoparticle (CLIO-NP) imaging. Materials and Methods: Female wild-type (n = 23) and myeloperoxidase (MPO) knock-out (n = 5) mice were infected with the Plasmodium berghei ANKA strain from May 2016 to July 2018. Seven healthy mice served as control animals. At a Rapid Murine Coma and Behavioral Scale (RMCBS) score of less than 15, mice underwent MRI at 9.4 T and received gadodiamide, MPOGd, or CLIO-NPs. T1-weighted MRI was used to assess MPO activity, and T2*-weighted MRI was used to track CLIO-NPs. Immunofluorescent staining and flow cytometric analyses characterized CLIO-NPs, MPO, endothelial cells, and leukocytes. An unpaired, two-tailed Student t test was used to compare groups; Spearman correlation analysis was used to determine the relationship of imaging parameters to clinical severity. Results: MPO-Gd enhancement occurred in inflammatory CM hotspots (olfactory bulb > rostral migratory stream > brainstem. cortex, P<.05 for all regions compared with control mice; mean olfactory bulb signal intensity ratio: 1.40 +/- 0.07 vs 0.96 +/- 0.01, P<.01). The enhancement was reduced in MPO knockout mice (mean signal intensity ratio at 60 minutes: 1.13 +/- 0.04 vs 1.40 +/- 0.07 in CM, P<.05). Blood-brain barrier compromise was suggested by parenchymal gadolinium enhancement, leukocyte recruitment, and endothelial activation. CLIO-NPs accumulated mainly intravascularly and at the vascular endothelium. CLIONPs were also found in the choroid plexus, indicating inflammation of the ventricular system. Blood-cerebrospinal fluid barrier breakdown showed correlation with brain swelling (r(2): 0.55, P<.01) and RMCBS score (r(2): 0.75, P<.001). Conclusion: Iron oxide nanoparticle imaging showed strong inflammatory involvement of the microvasculature in a murine model of cerebral malaria. Furthermore, bis-5-hydroxy-tryptamide-diethylenetriaminepentaacetate gadolinium imaging depicted parenchymal and intraventricular inflammation. This combined molecular imaging approach links vascular inflammation to breakdown of the blood-brain barrier and blood-cerebrospinal fluid barrier that correlate with global brain edema and disease severity. (c) RSNA, 2018

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.

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.