Role of the GLUT1 Glucose Transporter in Postnatal CNS Angiogenesis and Blood-Brain Barrier Integrity.

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.

Hemodynamic response to sensory stimulation in mice: Comparison between functional ultrasound and optoacoustic imaging.

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.

Pericyte hypoxia-inducible factor-1 (HIF-1) drives blood-brain barrier disruption and impacts acute ischemic stroke outcome.

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.

Retrieving neuronal orientations using 3D scanning SAXS and comparison with diffusion MRI.

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.

Inhibiting mGluR5 activity by AFQ056/Mavoglurant rescues circuit-specific functional connectivity in Fmr1 knockout mice.

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.

Early alterations in functional connectivity and white matter structure in a transgenic mouse model of cerebral amyloidosis.

Impairment of brain functional connectivity (FC) is thought to be an early event occurring in diseases with cerebral amyloidosis, such as Alzheimer's disease. Regions sustaining altered functional networks have been shown to colocalize with regions marked with amyloid plaques burden suggesting a strong link between FC and amyloidosis. Whether the decline in FC precedes amyloid plaque deposition or is a consequence thereof is currently unknown. The sequence of events during early stages of the disease is difficult to capture in humans due to the difficulties in providing an early diagnosis and also in view of the heterogeneity among patients. Transgenic mouse lines overexpressing amyloid precursor proteins develop cerebral amyloidosis and constitute an attractive model system for studying the relationship between plaque and functional changes. In this study, ArcAß transgenic and wild-type mice were imaged using resting-state fMRI methods across their life-span in a cross-sectional design to analyze changes in FC in relation to the pathology. Transgenic mice show compromised development of FC during the first months of postnatal life compared with wild-type animals, resulting in functional impairments that affect in particular the sensory-motor cortex already in preplaque stage. These functional alterations were accompanied by structural changes as reflected by reduced fractional anisotropy values, as derived from diffusion tensor imaging. Our results suggest cerebral amyloidosis in mice is preceded by impairment of neuronal networks and white matter structures. FC analysis in mice is an attractive tool for studying the implications of impaired neuronal networks in models of cerebral amyloid pathology.

The hemodynamic response to somatosensory stimulation in mice depends on the anesthetic used: Implications on analysis of mouse fMRI data.

In recent years, the number of functional MRI (fMRI) studies in mice has been rapidly increasing. Technological improvements provide the sensitivity required to match the high demands on spatial and temporal resolution and to analyze fast and small signal components of the fMRI response. Yet, the interpretation of mouse fMRI data largely relies on assumptions that were uncritically adopted from previous research in humans or rats. Here, we show based on a large dataset employing an innocuous electrical stimulation paradigm, that (1) the shape of the HRF shapes comprises significant transient signal components; correspondingly analysis procedures have to account for this dynamic nature and allow for variable response functions. (2) The effects of the anesthetics are crucial in determining the shape of the hemodynamic response function (HRF) and also influence the spatial specificity of BOLD signal. (3) The dominant systemic confounding contributions elicited by stimulus-evoked cardiovascular responses observed in mouse fMRI when applying block stimuli may be largely avoided by a milder event-related design applying a randomly spaced single pulse train (RSSPT). Thereby the spatial specificity of the fMRI response is largely retained. We conclude that the sensitivity, specificity and interpretability of stimulus-evoked BOLD signals in mice can be improved by combining appropriate stimulation paradigms with analysis procedures that include adapted HRFs.

Metabolic changes assessed by MRS accurately reflect brain function during drug-induced epilepsy in mice in contrast to fMRI-based hemodynamic readouts.

Functional proton magnetic resonance spectroscopy (1H-MRS) enables the non-invasive assessment of neural activity by measuring signals arising from endogenous metabolites in a time resolved manner. Proof-of-principle of this approach has been demonstrated in humans and rats; yet functional 1H-MRS has not been applied in mice so far, although it would be of considerable interest given the many genetically engineered models of neurological disorders established in this species only. Mouse 1H-MRS is challenging as the high demands on spatial resolution typically result in long data acquisition times not commensurable with functional studies. Here, we propose an approach based on spectroscopic imaging in combination with the acquisition of the free induction decay to maximize signal intensity. Highly resolved metabolite maps have been recorded from mouse brain with 12 min temporal resolution. This enabled monitoring of metabolic changes following the administration of bicuculline, a GABA-A receptor antagonist. Changes in levels of metabolites involved in energy metabolism (lactate and phosphocreatine) and neurotransmitters (glutamate) were investigated in a region-dependent manner and shown to scale with the bicuculline dose. GABAergic inhibition induced spectral changes characteristic for increased neurotransmitter turnover and oxidative stress. In contrast to metabolic readouts, BOLD and CBV fMRI responses did not scale with the bicuculline dose indicative of the failure of neurovascular coupling. Nevertheless fMRI measurements supported the notion of increased oxidative stress revealed by functional MRS. Hence, the combined analysis of metabolic and hemodynamic changes in response to stimulation provides complementary insight into processes associated with neural activity.

Optimization of anesthesia protocol for resting-state fMRI in mice based on differential effects of anesthetics on functional connectivity patterns.

Resting state-fMRI (rs-fMRI) in mice allows studying mechanisms underlying functional connectivity (FC) as well as alterations of FC occurring in murine models of neurological diseases. Mouse fMRI experiments are typically carried out under anesthesia to minimize animal movement and potential distress during examination. Yet, anesthesia inevitably affects FC patterns. Such effects have to be understood for proper interpretation of data. We have compared the influence of four commonly used anesthetics on rs-fMRI. Rs-fMRI data acquired under isoflurane, propofol, and urethane presented similar patterns when accounting for anesthesia depth. FC maps displayed bilateral correlation with respect to cortical seeds, but no significant inter-hemispheric striatal connectivity. In contrast, for medetomidine, we detected bilateral striatal but compromised inter-hemispheric cortical connectivity. The spatiotemporal patterns of the rs-fMRI signal have been rationalized considering anesthesia depth and pharmacodynamic properties of the anesthetics. Our results bridge the results from different studies from the burgeoning field of mouse rs-fMRI and offer a framework for understanding the influences of anesthetics on FC patterns. Utilizing this information, we suggest the combined use of medetomidine and isoflurane representing the two proposed classes of anesthetics; the combination of low doses of the two anesthetics retained strong correlations both within cortical and subcortical structures, without the potential seizure-inducing effects of medetomidine, rendering this regimen an attractive anesthesia for rs-fMRI in mice.

Specificity of stimulus-evoked fMRI responses in the mouse: the influence of systemic physiological changes associated with innocuous stimulation under four different anesthetics.

Functional magnetic resonance (fMRI) in mice has become an attractive tool for mechanistic studies, for characterizing models of human disease, and for evaluation of novel therapies. Yet, controlling the physiological state of mice is challenging, but nevertheless important as changes in cardiovascular parameters might affect the hemodynamic readout which constitutes the basics of the fMRI signal. In contrast to rats, fMRI studies in mice report less robust brain activation of rather widespread character to innocuous sensory stimulation. Anesthesia is known to influence the characteristics of the fMRI signal. To evaluate modulatory effects imposed by the anesthesia on stimulus-evoked fMRI responses, we compared blood oxygenation level dependent (BOLD) and cerebral blood volume (CBV) signal changes to electrical hindpaw stimulation using the four commonly used anesthetics isoflurane, medetomidine, propofol and urethane. fMRI measurements were complemented by assessing systemic physiological parameters throughout the experiment. Unilateral stimulation of the hindpaw elicited widespread fMRI responses in the mouse brain displaying a bilateral pattern irrespective of the anesthetic used. Analysis of magnitude and temporal profile of BOLD and CBV signals indicated anesthesia-specific modulation of cerebral hemodynamic responses and differences observed for the four anesthetics could be largely explained by their known effects on animal physiology. Strikingly, independent of the anesthetic used our results reveal that fMRI responses are influenced by stimulus-induced cardiovascular changes, which indicate an arousal response, even to innocuous stimulation. This may mask specific fMRI signal associated to the stimulus. Hence, studying the processing of peripheral input in mice using fMRI techniques constitutes a major challenge and adapted paradigms and/or alternative fMRI readouts should also be considered when studying sensory processing in mice.

Upregulation of axon guidance molecules in the adult central nervous system of Nogo-A knockout mice restricts neuronal growth and regeneration.

Adult central nervous system axons show restricted growth and regeneration properties after injury. One of the underlying mechanisms is the activation of the Nogo-A/Nogo receptor (NgR1) signaling pathway. Nogo-A knockout (KO) mice show enhanced regenerative growth in vivo, even though it is less pronounced than after acute antibody-mediated neutralization of Nogo-A. Residual inhibition may involve a compensatory component. By mRNA expression profiling and immunoblots we show increased expression of several members of the Ephrin/Eph and Semaphorin/Plexin families of axon guidance molecules, e.g. EphrinA3 and EphA4, in the intact spinal cord of adult Nogo-A KO vs. wild-type (WT) mice. EphrinA3 inhibits neurite outgrowth of EphA4-positive neurons in vitro. In addition, EphrinA3 KO myelin extracts are less growth-inhibitory than WT but more than Nogo-A KO myelin extracts. EphA4 KO cortical neurons show decreased growth inhibition on Nogo-A KO myelin as compared with WT neurons, supporting increased EphA4-mediated growth inhibition in Nogo-A KO mice. Consistently, in vivo, Nogo-A/EphA4 double KO mice show increased axonal sprouting and regeneration after spinal cord injury as compared with EphA4 KO mice. Our results reveal the upregulation of developmental axon guidance cues following constitutive Nogo-A deletion, e.g. the EphrinA3/EphA4 ligand/receptor pair, and support their role in restricting neurite outgrowth in the absence of Nogo-A.

Neuronal Nogo-A regulates neurite fasciculation, branching and extension in the developing nervous system.

Wiring of the nervous system is a multi-step process involving complex interactions of the growing fibre with its tissue environment and with neighbouring fibres. Nogo-A is a membrane protein enriched in the adult central nervous system (CNS) myelin, where it restricts the capacity of axons to grow and regenerate after injury. During development, Nogo-A is also expressed by neurons but its function in this cell type is poorly known. Here, we show that neutralization of neuronal Nogo-A or Nogo-A gene ablation (KO) leads to longer neurites, increased fasciculation, and decreased branching of cultured dorsal root ganglion neurons. The same effects are seen with antibodies against the Nogo receptor complex components NgR and Lingo1, or by blocking the downstream effector Rho kinase (ROCK). In the chicken embryo, in ovo injection of anti-Nogo-A antibodies leads to aberrant innervation of the hindlimb. Genetic ablation of Nogo-A causes increased fasciculation and reduced branching of peripheral nerves in Nogo-A KO mouse embryos. Thus, Nogo-A is a developmental neurite growth regulatory factor with a role as a negative regulator of axon-axon adhesion and growth, and as a facilitator of neurite branching.

Imaging crossing fibers in mouse, pig, monkey, and human brain using small-angle X-ray scattering.

Myelinated axons (nerve fibers) efficiently transmit signals throughout the brain via action potentials. Multiple methods that are sensitive to axon orientations, from microscopy to magnetic resonance imaging, aim to reconstruct the brain's structural connectome. As billions of nerve fibers traverse the brain with various possible geometries at each point, resolving fiber crossings is necessary to generate accurate structural connectivity maps. However, doing so with specificity is a challenging task because signals originating from oriented fibers can be influenced by brain (micro)structures unrelated to myelinated axons. X-ray scattering can specifically probe myelinated axons due to the periodicity of the myelin sheath, which yields distinct peaks in the scattering pattern. Here, we show that small-angle X-ray scattering (SAXS) can be used to detect myelinated, axon-specific fiber crossings. We first demonstrate the capability using strips of human corpus callosum to create artificial double- and triple-crossing fiber geometries, and we then apply the method in mouse, pig, vervet monkey, and human brains. We compare results to polarized light imaging (3D-PLI), tracer experiments, and to outputs from diffusion MRI that sometimes fails to detect crossings. Given its specificity, capability of 3-dimensional sampling and high resolution, SAXS could serve as a ground truth for validating fiber orientations derived using diffusion MRI as well as microscopy-based methods. STATEMENT OF SIGNIFICANCE: To study how the nerve fibers in our brain are interconnected, scientists need to visualize their trajectories, which often cross one another. Here, we show the unique capacity of small-angle X-ray scattering (SAXS) to study these fiber crossings without use of labeling, taking advantage of SAXS's specificity to myelin - the insulating sheath that is wrapped around nerve fibers. We use SAXS to detect double and triple crossing fibers and unveil intricate crossings in mouse, pig, vervet monkey, and human brains. This non-destructive method can uncover complex fiber trajectories and validate other less specific imaging methods (e.g., MRI or microscopy), towards accurate mapping of neuronal connectivity in the animal and human brain.

Hybrid fiber optic-fMRI for multimodal cell-specific recording and manipulation of neural activity in rodents.

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.

Systematic Review: Anesthetic Protocols and Management as Confounders in Rodent Blood Oxygen Level Dependent Functional Magnetic Resonance Imaging (BOLD fMRI)-Part B: Effects of Anesthetic Agents, Doses and Timing.

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.

Nanostructure-specific X-ray tomography reveals myelin levels, integrity and axon orientations in mouse and human nervous tissue.

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.

Systematic Review: Anaesthetic Protocols and Management as Confounders in Rodent Blood Oxygen Level Dependent Functional Magnetic Resonance Imaging (BOLD fMRI)-Part A: Effects of Changes in Physiological Parameters.

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.

Functional spectroscopic imaging reveals specificity of glutamate response in mouse brain to peripheral sensory stimulation.

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.

Early reduced behavioral activity induced by large strokes affects the efficiency of enriched environment in rats.

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.

Fiber-optic implant for simultaneous fluorescence-based calcium recordings and BOLD fMRI in mice.

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.