Small-angle X-ray scattering datasets for resolving crossing fibers (myelinated neuronal axon bundles), as described in "Imaging crossing fibers in mouse, pig, monkey, and human brain using small-angle X-ray scattering" deposited in bioRxiv: https://doi.org/10.1101/2022.09.30.510198

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.

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.

Significance: Multiscale imaging holds particular relevance to neuroscience, where it helps integrate the cellular and molecular biological scale, which is most accessible to interventions, with holistic organ-level evaluations, most relevant with respect to function. Being inextricably interdisciplinary, multiscale imaging benefits substantially from incremental technology adoption, and a detailed overview of the state-of-the-art is vital to an informed application of imaging methods. Aim: In this article, we lay out the background and methodological aspects of multimodal approaches combining functional magnetic resonance imaging (fMRI) with simultaneous optical measurement or stimulation. Approach: We focus on optical techniques as these allow, in conjunction with genetically encoded proteins (e.g. calcium indicators or optical signal transducers), unprecedented read-out and control specificity for individual cell-types during fMRI experiments, while leveraging non-interfering modalities. Results: A variety of different solutions for optical/fMRI methods has been reported ranging from bulk fluorescence recordings via fiber photometry to high resolution microscopy. In particular, the plethora of optogenetic tools has enabled the transformation of stimulus-evoked fMRI into a cell biological interrogation method. We discuss the capabilities and limitations of these genetically encoded molecular tools in the study of brain phenomena of great methodological and neuropsychiatric interest-such as neurovascular coupling (NVC) and neuronal network mapping. We provide a methodological description of this interdisciplinary field of study, and focus in particular on the limitations of the widely used blood oxygen level dependent (BOLD) signal and how multimodal readouts can shed light on the contributions arising from neurons, astrocytes, or the vasculature. Conclusion: We conclude that information from multiple signaling pathways must be incorporated in future forward models of the BOLD response to prevent erroneous conclusions when using fMRI as a surrogate measure for neural activity. Further, we highlight the potential of direct neuronal stimulation via genetically defined brain networks towards advancing neurophysiological understanding and better estimating effective connectivity. (C) The Authors. Published by SPIE under a Creative Commons Attribution 4.0 International License.

Intense efforts are underway to develop functional imaging modalities for capturing brain activity at the whole organ scale with high spatial and temporal resolution. Functional optoacoustic (fOA) imaging is emerging as a new tool to monitor multiple hemodynamic parameters across the mouse brain, but its sound validation against other neuroimaging modalities is often lacking. Here we investigate mouse brain responses to peripheral sensory stimulation using both fOA and functional ultrasound (fUS) imaging. The two modalities operate under similar spatio-temporal resolution regime, with a potential to provide synergistic and complementary hemodynamic readouts. Specific contralateral activation was observed with sub-millimeter spatial resolution with both methods. Sensitivity to hemodynamic activity was found to be on comparable levels, with the strongest responses obtained in the oxygenated hemoglobin channel of fOA. While the techniques attained highly correlated hemodynamic responses, the differential fOA readings of oxygenated and deoxygenated haemoglobin provided complementary information to the blood flow contrast of fUS. The multi-modal approach may thus emerge as a powerful tool providing new insights into brain function, complementing our current knowledge generated with well-established neuroimaging methods.

Myelin insulates neuronal axons and enables fast signal transmission, constituting a key component of brain development, aging and disease. Yet, myelin-specific imaging of macroscopic samples remains a challenge. Here, we exploit myelin's nanostructural periodicity, and use small-angle X-ray scattering tensor tomography (SAXS-TT) to simultaneously quantify myelin levels, nanostructural integrity and axon orientations in nervous tissue. Proof-of-principle is demonstrated in whole mouse brain, mouse spinal cord and human white and gray matter samples. Outcomes are validated by 2D/3D histology and compared to MRI measurements sensitive to myelin and axon orientations. Specificity to nanostructure is exemplified by concomitantly imaging different myelin types with distinct periodicities. Finally, we illustrate the method's sensitivity towards myelin-related diseases by quantifying myelin alterations in dysmyelinated mouse brain. This non-destructive, stain-free molecular imaging approach enables quantitative studies of myelination within and across samples during development, aging, disease and treatment, and is applicable to other ordered biomolecules or nanostructures.

In rodent models the use of functional magnetic resonance imaging (fMRI) under anesthesia is common. The anesthetic protocol might influence fMRI readouts either directly or via changes in physiological parameters. As long as those factors cannot be objectively quantified, the scientific validity of fMRI in rodents is impaired. In the present systematic review, literature analyzing in rats and mice the influence of anesthesia regimes and concurrent physiological functions on blood oxygen level dependent (BOLD) fMRI results was investigated. Studies from four databases that were searched were selected following pre-defined criteria. Two separate articles publish the results; the herewith presented article includes the analyses of 83 studies. Most studies found differences in BOLD fMRI readouts with different anesthesia drugs and dose rates, time points of imaging or when awake status was compared to anesthetized animals. To obtain scientifically valid, reproducible results from rodent fMRI studies, stable levels of anesthesia with agents suitable for the model under investigation as well as known and objectively quantifiable effects on readouts are, thus, mandatory. Further studies should establish dose ranges for standardized anesthetic protocols and determine time windows for imaging during which influence of anesthesia on readout is objectively quantifiable

Pericytes play essential roles in blood-brain barrier integrity and their dysfunction is implicated in neurological disorders such as stroke although the underlying mechanisms remain unknown. Hypoxia-inducible factor-1 (HIF-1), a master regulator of injury responses, has divergent roles in different cells especially during stress scenarios. On one hand HIF-1 is neuroprotective but on the other it induces vascular permeability. Since pericytes are critical for barrier stability, we asked if pericyte HIF-1 signaling impacts barrier integrity and injury severity in a mouse model of ischemic stroke. We show that pericyte HIF-1 loss of function (LoF) diminishes ischemic damage and barrier permeability at 3 days reperfusion. HIF-1 deficiency preserved barrier integrity by reducing pericyte death thereby maintaining vessel coverage and junctional protein organization, and suppressing vascular remodeling. Importantly, considerable improvements in sensorimotor function were observed in HIF-1 LoF mice indicating that better vascular functionality post stroke improves outcome. Thus, boosting vascular integrity by inhibiting pericytic HIF-1 activation and/or increasing pericyte survival may be a lucrative option to accelerate recovery after severe brain injury.

Background: To understand brain function in health and disease, functional magnetic resonance imaging (fMRI) is widely used in rodent models. Because animals need to be immobilised for image acquisition, fMRI is commonly performed under anaesthesia. The choice of anaesthetic protocols and may affect fMRI readouts, either directly or via changing physiological balance, and thereby threaten the scientific validity of fMRI in rodents. Methods: The present study systematically reviewed the literature investigating the influence of different anaesthesia regimes and changes in physiological parameters as confounders of blood oxygen level dependent (BOLD) fMRI in rats and mice. Four databases were searched, studies selected according to pre-defined criteria, and risk of bias assessed for each study. Results are reported in two separate articles; this part of the review focuses on effects of changes in physiological parameters. Results: A total of 121 publications was included, of which 49 addressed effects of changes in physiological parameters. Risk of bias was high in all included studies. Blood oxygenation [arterial partial pressure of oxygen (paO2)], ventilation [arterial partial pressure of carbon dioxide (paCO2)] and arterial blood pressure affected BOLD fMRI readouts across various experimental paradigms. Conclusions: Blood oxygenation, ventilation and arterial blood pressure should be monitored and maintained at stable physiological levels throughout experiments. Appropriate anaesthetic management and monitoring are crucial to obtain scientifically valid, reproducible results from fMRI studies in rodent models.

RATIONALE:Endothelial cells (ECs) are highly glycolytic and generate the majority of their energy via the breakdown of glucose to lactate. At the same time, a main role of ECs is to allow the transport of glucose to the surrounding tissues. GLUT1 (glucose transporter isoform 1/Slc2a1) is highly expressed in ECs of the central nervous system (CNS) and is often implicated in blood-brain barrier (BBB) dysfunction, but whether and how GLUT1 controls EC metabolism and function is poorly understood.OBJECTIVE:We evaluated the role of GLUT1 in endothelial metabolism and function during postnatal CNS development as well as at the adult BBB.METHODS AND RESULTS:Inhibition of GLUT1 decreases EC glucose uptake and glycolysis, leading to energy depletion and the activation of the cellular energy sensor AMPK (AMP-activated protein kinase), and decreases EC proliferation without affecting migration. Deletion of GLUT1 from the developing postnatal retinal endothelium reduces retinal EC proliferation and lowers vascular outgrowth, without affecting the number of tip cells. In contrast, in the brain, we observed a lower number of tip cells in addition to reduced brain EC proliferation, indicating that within the CNS, organotypic differences in EC metabolism exist. Interestingly, when ECs become quiescent, endothelial glycolysis is repressed, and GLUT1 expression increases in a Notch-dependent fashion. GLUT1 deletion from quiescent adult ECs leads to severe seizures, accompanied by neuronal loss and CNS inflammation. Strikingly, this does not coincide with BBB leakiness, altered expression of genes crucial for BBB barrier functioning nor reduced vascular function. Instead, we found a selective activation of inflammatory and extracellular matrix related gene sets.CONCLUSIONS:GLUT1 is the main glucose transporter in ECs and becomes uncoupled from glycolysis during quiescence in a Notch-dependent manner. It is crucial for developmental CNS angiogenesis and adult CNS homeostasis but does not affect BBB barrier function.

While diffusion MRI (dMRI) is currently the method of choice to non-invasively probe tissue 14 microstructure and study structural connectivity in the brain, its spatial resolution is limited and its 15 results need structural validation. Current ex vivo methods employed to provide 3D fiber orientations 16 have limitations, including tissue-distorting sample preparation, small field of view or inability to 17 quantify 3D fiber orientation distributions. 3D fiber orientation in tissue sections can be obtained 18 from 3D scanning small-angle X-ray scattering (3D sSAXS) by analyzing the anisotropy of scattering 19 signals. Here we adapt the 3D sSAXS method for use in brain tissue, exploiting the high sensitivity 20 of the SAXS signal to the ordered molecular structure of myelin. We extend the characterization of 21 anisotropy from vectors to tensors, employ the Funk-Radon-Transform for converting scattering 22 information to real space fiber orientations, and demonstrate the feasibility of the method in thin 23 sections of mouse brain with minimal sample preparation. We obtain a second rank tensor 24 representing the fiber orientation distribution function (fODF) for every voxel, thereby generating 25 fODF maps. Finally, we illustrate the potential of 3D sSAXS by comparing the result with diffusion 26 MRI fiber orientations in the same mouse brain. We show a remarkably good correspondence, 27 considering the orthogonality of the two methods, i.e. the different physical processes underlying 28 the two signals. 3D sSAXS can serve as validation method for microstructural MRI, and can provide 29 novel microstructural insights for the nervous system, given the method’s orthogonality to dMRI, 30 high sensitivity to myelin sheath’s orientation and abundance, and the possibility to extract myelin31 specific signal and to perform micrometer-resolution scanning. 32

Non-invasive investigation of physiological changes and metabolic events associated with brain activity in mice constitutes a major challenge. Conventionally, fMRI assesses neuronal activity by evaluating activity-evoked local changes in blood oxygenation levels (BOLD). In isoflurane-anaethetized mice, however, we found that BOLD signal changes during paw stimulation appear to be dominated by arousal responses even when using innocuous stimuli. Widespread responses involving both hemispheres have been observed in response to unilateral stimulation. MRS allows probing metabolic changes associated with neuronal activation and provides a complementary readout to BOLD fMRI for investigating brain activity. In this study we evaluated the sensitivity of a free induction decay (FID) based spectroscopic imaging (MRSI) protocol for the measurement of alterations in glutamate levels elicited by unilateral electrical paw stimulation at different current amplitudes. Coronal MRSI maps of glutamate distribution with 17 × 17 voxels of 1 µl volume have been recorded with a temporal resolution of 12 min. Significant region-specific increases in glutamate levels have been observed in the contralateral but not in the ispiateral S1 somatosensory cortex upon stimulation. The amplitude of glutamate changes increased in a dose-dependent manner with the stimulus amplitude. The study demonstrates feasibility of functional MRSI in mice for studying activity-evoked glutamate changes in a temporo-spatially resolved manner.

Previous work has demonstrated that neuroimaging biomarkers which capture functional connectivity of the brain can be used to define a specific and robust endophenotype in Fmr1-/y mice, a well-established animal model of human Fragile-X Syndrome (FXS). However, it is currently unknown whether this macroscopic measure of brain connectivity is sufficiently sensitive to reliably detect changes caused by pharmacological interventions. Here we inhibited the activity of the metabotropic glutamate receptor-5 (mGluR5) using AFQ056/Mavoglurant, a drug that is assumed to normalize excitatory/inhibitory neural signaling imbalances in FXS. We employed resting-state-fMRI (rs-fMRI) and diffusion-weighted imaging (DWI) to test whether Mavoglurant re-established brain connectivity - at least partly - within some of the affected circuits in Fmr1-/y mice that are related to social behavior deficits. In line with previous findings, we observed that Fmr1-/y mice exhibited impaired social interaction, reduced connectivity in three main functional networks and altered network topology. At the group level, Mavoglurant did neither rescue abnormal social behavioral nor white matter abnormalities; however, for some, but not all of these circuits Mavoglurant had a genotype-specific effect of restoring functional connectivity. These results show that rs-fMRI connectivity is sufficiently sensitive to pick up system-level changes after the pharmacological inhibition of mGluR5 activity. However, our results also show that the effects of Mavoglurant are confined to specific networks suggesting that behavioral benefits might be restricted to narrow functional domains.

While diffusion MRI (dMRI) is currently the method of choice to non-invasively probe tissue microstructure and study structural connectivity in the brain, its spatial resolution is limited and its results need structural validation. Current ex vivo methods employed to provide 3D fiber orientations have limitations, including tissue-distorting sample preparation, small field of view or inability to quantify 3D fiber orientation distributions. 3D fiber orientation in tissue sections can be obtained from 3D scanning small-angle X-ray scattering (3D sSAXS) by analyzing the anisotropy of scattering signals. Here we adapt the 3D sSAXS method for use in brain tissue, exploiting the high sensitivity of the SAXS signal to the ordered molecular structure of myelin. We extend the characterization of anisotropy from vectors to tensors, employ the Funk-Radon-Transform for converting scattering information to real space fiber orientations, and demonstrate the feasibility of the method in thin sections of mouse brain with minimal sample preparation. We obtain a second rank tensor representing the fiber orientation distribution function (fODF) for every voxel, thereby generating fODF maps. Finally, we illustrate the potential of 3D sSAXS by comparing the result with diffusion MRI fiber orientations in the same mouse brain. We show a remarkably good correspondence, considering the orthogonality of the two methods, i.e. the different physical processes underlying the two signals. 3D sSAXS can serve as validation method for microstructural MRI, and can provide novel microstructural insights for the nervous system, given the method's orthogonality to dMRI, high sensitivity to myelin sheath's orientation and abundance, and the possibility to extract myelin-specific signal and to perform micrometer-resolution scanning.

Despite the growing popularity of blood oxygen level-dependent (BOLD) functional MRI (fMRI), understanding of its underlying principles is still limited. This protocol describes a technique for simultaneous measurement of neural activity using fluorescent calcium indicators together with the corresponding hemodynamic BOLD fMRI response in the mouse brain. Our early work using small-molecule fluorophores in rats gave encouraging results but was limited to acute measurements using synthetic dyes. Our latest procedure combines fMRI with optical detection of cell-type-specific virally delivered GCaMP6, a genetically encoded calcium indicator (GECI). GCaMP6 fluorescence, which increases upon calcium binding, is collected by a chronically implanted optical fiber, allowing longitudinal studies in mice. The chronic implant, placed horizontally on the skull, has an angulated tip that reflects light into the brain and is connected via fiber optics to a remote optical setup. The technique allows access to the neocortex and does not require adaptations of commercial MRI hardware. The hybrid approach permits fiber-optic calcium recordings with simultaneous artifact-free BOLD fMRI with full brain coverage and 1-s temporal resolution using standard gradient-echo echo-planar imaging (GE-EPI) sequences. The method provides robust, cell-type-specific readouts to link neural activity to BOLD signals, as emonstrated for task-free ('resting-state') conditions and in response to hind-paw stimulation. These results highlight the power of fiber photometry combined with fMRI, which we aim to further advance in this protocol. The approach can be easily adapted to study other molecular processes using suitable fluorescent indicators.

The majority of stroke patients develop post-stroke fatigue, a symptom which impairs motivation and diminishes the success of rehabilitative interventions. We show that large cortical strokes acutely reduce activity levels in rats for 1-2 weeks as a physiological response paralleled by signs of systemic inflammation. Rats were exposed early (1-2 weeks) or late (3-4 weeks after stroke) to an individually monitored enriched environment to stimulate self-controlled high-intensity sensorimotor training. A group of animals received Anti-Nogo antibodies for the first two weeks after stroke, a neuronal growth promoting immunotherapy already in clinical trials. Early exposure to the enriched environment resulted in poor outcome: Training intensity was correlated to enhanced systemic inflammation and functional impairment. In contrast, animals starting intense sensorimotor training two weeks after stroke preceded by the immunotherapy revealed better recovery with functional outcome positively correlated to the training intensity and the extent of re-innervation of the stroke denervated cervical hemi-cord. Our results suggest stroke-induced fatigue as a biological purposeful reaction of the organism during neuronal remodeling, enabling new circuit formation which will then be stabilized or pruned in the subsequent rehabilitative training phase. However, intense training too early may lead to wrong connections and is thus less effective.

Previously, we reported widespread bilateral increases in stimulus-evoked functional magnetic resonance imaging signals in mouse brain to unilateral sensory paw stimulation. We attributed the pattern to arousal-related cardiovascular changes overruling cerebral autoregulation thereby masking specific signal changes elicited by local neuronal activity. To rule out the possibility that interhemispheric neuronal communication might contribute to bilateral functional magnetic resonance imaging responses, we compared stimulus-evoked functional magnetic resonance imaging responses to unilateral hindpaw stimulation in acallosal I/LnJ, C57BL/6, and BALB/c mice. We found bilateral blood-oxygenation-level dependent signal changes in all three strains, ruling out a dominant contribution of transcallosal communication as reason for bilaterality. Analysis of functional connectivity derived from resting-state functional magnetic resonance imaging, revealed that bilateral cortical functional connectivity is largely abolished in I/LnJ animals. Cortical functional connectivity in all strains correlated with structural connectivity in corpus callosum as revealed by diffusion tensor imaging. Given the profound influence of systemic hemodynamics on stimulus-evoked functional magnetic resonance imaging outcomes, we evaluated whether functional connectivity data might be affected by cerebrovascular parameters, i.e. baseline cerebral blood volume, vascular reactivity, and reserve. We found that effects of cerebral hemodynamics on functional connectivity are largely outweighed by dominating contributions of structural connectivity. In contrast, contributions of transcallosal interhemispheric communication to the occurrence of ipsilateral functional magnetic resonance imaging response of equal amplitude to unilateral stimuli seem negligible.

fMRI studies in mice typically require the use of anesthetics. Yet, it is known that anesthesia alters responses to stimuli or functional networks at rest. In this work, we have used Dual Regression analysis Network Modeling to investigate the effects of two commonly used anesthetics, isoflurane and medetomidine, on rs-fMRI derived functional networks, and in particular to what extent anesthesia affected the interaction within and between these networks. Experimental data have been used from a previous study (Grandjean et al., 2014). We applied multivariate ICA analysis and Dual Regression to infer the differences in functional connectivity between isoflurane- and medetomidine-anesthetized mice. Further network analysis was performed to investigate within- and between-network connectivity differences between these anesthetic regimens. The results revealed five major networks in the mouse brain: lateral cortical, associative cortical, default mode, subcortical, and thalamic network. The anesthesia regime had a profound effect both on within- and between-network interactions. Under isoflurane anesthesia predominantly intra- and inter-cortical interactions have been observed, with only minor interactions involving subcortical structures and in particular attenuated cortico-thalamic connectivity. In contrast, medetomidine-anesthetized mice displayed subcortical functional connectivity including interactions between cortical and thalamic ICA components. Combining the two anesthetics at low dose resulted in network interaction that constituted the superposition of the interaction observed for each anesthetic alone. The study demonstrated that network modeling is a promising tool for analyzing the brain functional architecture in mice and comparing alterations therein caused by different physiological or pathological states. Understanding the differential effects of anesthetics on brain networks and their interaction is essential when interpreting fMRI data recorded under specific physiological and pathological conditions.

The dopamine D-2 receptor (D2R) couples to inhibitory G(i/o) proteins and is targeted by antipsychotic and antiparkinsonian drugs. Beta-arrestin2 binds to the intracellular regions of the agonist-occupied D2R to terminate G protein activation and promote internalization, but also to initiate downstream signaling cascades which have been implicated in psychosis. Functional magnetic resonance imaging (fMRI) has proven valuable for measuring dopamine receptor-mediated changes in neuronal activity, and might enable beta-arrestin2 function to be studied in vivo.The present study examined fMRI blood oxygenation level dependent (BOLD) signal changes elicited by a dopamine agonist in wild-type (WT) and beta-arrestin2 knockout (KO) mice, to investigate whether genetic deletion of beta-arrestin2 prolongs or otherwise modifies D2R-dependent responses.fMRI BOLD data were acquired on a 9.4 T system. During scans, animals received 0.2 mg/kg apomorphine, i.v. In a subset of experiments, animals were pretreated with 2 mg/kg of the D2R antagonist, eticlopride.Following apomorphine administration, BOLD signal decreases were observed in caudate/putamen of WT and KO animals. The time course of response decay in caudate/putamen was significantly slower in KO vs. WT animals. In cingulate cortex, an initial BOLD signal decrease was followed by a positive response component in WT but not in KO animals. Eticlopride pretreatment significantly reduced apomorphine-induced BOLD signal changes.The prolonged striatal response decay rates in KO animals might reflect impaired D2R desensitization, consistent with the known function of beta-arrestin2. Furthermore, the apomorphine-induced positive response component in cingulate cortex may depend on beta-arrestin2 signaling downstream of D2R.

Functional magnetic resonance imaging (fMRI) has revolutionized neuroscience by opening a unique window that allows neurocircuitry function and pathological alterations to be probed non-invasively across brain disorders. Here we report a novel sustainable anesthesia procedure for small animal neuroimaging that overcomes shortcomings of anesthetics commonly used in rodent fMRI. The significantly improved preservation of cerebrovascular dynamics enhances sensitivity to neural activity changes for which it serves as a proxy in fMRI readouts. Excellent cross-species/strain applicability provides coherence among preclinical findings and is expected to improve translation to clinical fMRI investigations. The novel anesthesia procedure based on the GABAergic anesthetic etomidate was extensively validated in fMRI studies conducted in a range of genetically engineered rodent models of autism and strains commonly used for transgenic manipulations. Etomidate proved effective, yielded long-term stable physiology with basal cerebral blood flow of ~0.5 ml/g/min and full recovery. Cerebrovascular responsiveness of up to 180% was maintained as demonstrated with perfusion- and BOLD-based fMRI upon hypercapnic, pharmacological and sensory stimulation. Hence, etomidate lends itself as an anesthetic-of-choice for translational neuroimaging studies across rodent models of brain disorders.