fMRI mapping of the visual system in the mouse brain with interleaved snapshot GE-EPI

The use of functional magnetic resonance imaging (fMRI) in mice is increasingly prevalent, providing a means to non-invasively characterise functional abnormalities associated with genetic models of human diseases. The predominant stimulus used in task-based fMRI in the mouse is electrical stimulation of the paw. Task-based fMRI in mice using visual stimuli remains underexplored, despite visual stimuli being common in human fMRI studies. In this study, we map the mouse brain visual system with BOLD measurements at 9.4T using flashing light stimuli with medetomidine anaesthesia. BOLD responses were observed in the lateral geniculate nucleus, the superior colliculus and the primary visual area of the cortex, and were modulated by the flashing frequency, diffuse vs focussed light and stimulus context. Negative BOLD responses were measured in the visual cortex at 10Hz flashing frequency; but turned positive below 5Hz. In addition, the use of interleaved snapshot GE-EPI improved fMRI image quality without diminishing the temporal contrast-noise-ratio. Taken together, this work demonstrates a novel methodological protocol in which the mouse brain visual system can be non-invasively investigated using BOLD fMRI

Investigation of robust visual reaction and functional connectivity in the rat brain induced by rocuronium bromide with functional MRI

Functional magnetic resonance imaging (fMRI) has been used extensively to understand the brain function of a wide range of neurological and psychiatric disorders. When applied to animal studies, anesthesia is always used to reduce the movement of the animal and also reduce the impacts on the results of fMRI. Several awake models have been proposed by applying physical animal movement restrictions. However, restraining devices were designed for individual subject which limits the promotion of fMRI in awake animals. Here, a clinical muscle relaxant rocuronium bromide (RB) was introduced to restrain the animal in fMRI scanning time. The fMRI reactions of the animal induced with RB and the other two commonly used anesthesia protocols were investigated. The results of the fMRI showed that there were increased functional connectivity and well-round visual responses in the RB induced state. Furthermore, significant BOLD signal changes were found in the cortex and thalamus regions when the animal revived from isoflurane, which should be essential to further understand the effects of anesthesia on the brain. Keywords: Rocuronium bromide, isoflurane, animal anesthesia, fMRI, visual stimulation, resting stat

Effects of Hemodynamic Response Function Selection on Rat fMRI Statistical Analyses

The selection of the appropriate hemodynamic response function (HRF) for signal modeling in functional magnetic resonance imaging (fMRI) is important. Although the use of the boxcar-shaped hemodynamic response function (BHRF) and canonical hemodynamic response (CHRF) has gained increasing popularity in rodent fMRI studies, whether the selected HRF affects the results of rodent fMRI has not been fully elucidated. Here we investigated the signal change and t-statistic sensitivities of BHRF, CHRF, and impulse response function (IRF). The effect of HRF selection on different tasks was analyzed by using data collected from two groups of rats receiving either 3 mA whisker pad or 3 mA forepaw electrical stimulations (n = 10 for each group). Under whisker pad stimulation with large blood-oxygen-level dependent (BOLD) signal change (4.31 ± 0.42%), BHRF significantly underestimated signal changes (P < 0.001) and t-statistics (P < 0.001) compared with CHRF or IRF. CHRF and IRF did not provide significantly different t-statistics (P > 0.05). Under forepaw stimulation with small BOLD signal change (1.71 ± 0.34%), different HRFs provided insignificantly different t-statistics (P > 0.05). Therefore, the selected HRF can influence data analysis in rodent fMRI experiments with large BOLD responses but not in those with small BOLD responses

Functional magnetic resonance imaging of the mouse brain

Functional magnetic resonance imaging (fMRI) measuring a blood-oxygen-level dependent (BOLD) signal is the most commonly used neuroimaging tool to understand brain function in humans. As mouse models are one of the most commonly used neuroscience experimental models, and with the advent of transgenic mouse models of neurodegenerative pathologies, there has been an increasing push in recent years to apply fMRI techniques to the mouse brain. This thesis focuses on the development and implementation of mouse brain fMRI techniques, in particular to describe the mouse visual system. Multiple studies in the literature have noted several technical challenges in mouse fMRI. In this work I have developed methods which go some way to reducing the impact of these issues, and I record robust and reliable haemodynamic-driven signal responses to visual stimuli in mouse brain regions specific to visual processing. I then developed increasingly complex visual stimuli, approaching the level of complexity used in electrophysiology studies of the mouse visual system, despite the geometric and magnetic field constraints of using a 9.4T pre-clinical MRI scanner. I have also applied a novel technique for measuring high-temporal resolution BOLD responses in the mouse superior colliculus, and I used this data to improve statistical parametric mapping of mouse brain BOLD responses. I also describe the first application of dynamic causal modelling to mouse fMRI data, characterising effective connectivity in the mouse brain visual system. This thesis makes significant contributions to the reverse translation of fMRI to the mouse brain, closing the gap between invasive electrophysiological measurements in the mouse brain and non-invasive fMRI measurements in the human brain

Generation of a whole-brain hemodynamic response function and sex-specific differences in cerebral processing of mechano-sensation in mice detected by BOLD fMRI

BOLD fMRI has become a prevalent method to study cerebral sensory processing in rodent disease models, including pain and mechanical hypersensitivity. fMRI data analysis is frequently combined with a general-linear-model (GLM) -based analysis, which uses the convolution of a hemodynamic response function (HRF) with the stimulus paradigm. However, several studies indicated that the HRF differs across species, sexes, brain structures, and experimental factors, including stimulation modalities or anesthesia, and hence might strongly affect the outcome of BOLD analyzes. While considerable work has been done in humans and rats to understand the HRF, much less is known in mice. As a prerequisite to investigate mechano-sensory processing and BOLD fMRI data in male and female mice, we (1) designed a rotating stimulator that allows application of two different mechanical modalities, including innocuous von Frey and noxious pinprick stimuli and (2) determined and statistically compared HRFs across 30 brain structures and experimental conditions, including sex and, stimulus modalities. We found that mechanical stimulation lead to brain-wide BOLD signal changes thereby allowing extraction of HRFs from multiple brain structures. However, we did not find differences in HRFs across all brain structures and experimental conditions. Hence, we computed a whole-brain mouse HRF, which is based on 88 functional scans from 30 mice. A comparison of this mouse-specific HRF with our previously reported rat-derived HRF showed significantly slower kinetics in mice. Finally, we detected pronounced differences in cerebral BOLD activation between male and female mice with mechanical stimulation, thereby exposing divergent processing of noxious and innocuous stimuli in both sexes

MP-PCA denoising of fMRI time-series data can lead to artificial activation "spreading"

MP-PCA denoising has become the method of choice for denoising in MRI since it provides an objective threshold to separate the desired signal from unwanted thermal noise components. In rodents, thermal noise in the coils is an important source of noise that can reduce the accuracy of activation mapping in fMRI. Further confounding this problem, vendor data often contains zero-filling and other effects that may violate MP-PCA assumptions. Here, we develop an approach to denoise vendor data and assess activation "spreading" caused by MP-PCA denoising in rodent task-based fMRI data. Data was obtained from N = 3 mice using conventional multislice and ultrafast acquisitions (1 s and 50 ms temporal resolution, respectively), during visual stimulation. MP-PCA denoising produced SNR gains of 64% and 39% and Fourier spectral amplitude (FSA) increases in BOLD maps of 9% and 7% for multislice and ultrafast data, respectively, when using a small [2 2] denoising window. Larger windows provided higher SNR and FSA gains with increased spatial extent of activation that may or may not represent real activation. Simulations showed that MP-PCA denoising causes activation "spreading" with an increase in false positive rate and smoother functional maps due to local "bleeding" of principal components, and that the optimal denoising window for improved specificity of functional mapping, based on Dice score calculations, depends on the data's tSNR and functional CNR. This "spreading" effect applies also to another recently proposed low-rank denoising method (NORDIC). Our results bode well for dramatically enhancing spatial and/or temporal resolution in future fMRI work, while taking into account the sensitivity/specificity trade-offs of low-rank denoising methods

The (un)conscious mouse as a model for human brain functions: key principles of anesthesia and their impact on translational neuroimaging

In recent years, technical and procedural advances have brought functional magnetic resonance imaging (fMRI) to the field of murine neuroscience. Due to its unique capacity to measure functional activity non-invasively, across the entire brain, fMRI allows for the direct comparison of large-scale murine and human brain functions. This opens an avenue for bidirectional translational strategies to address fundamental questions ranging from neurological disorders to the nature of consciousness. The key challenges of murine fMRI are: (1) to generate and maintain functional brain states that approximate those of calm and relaxed human volunteers, while (2) preserving neurovascular coupling and physiological baseline conditions. Low-dose anesthetic protocols are commonly applied in murine functional brain studies to prevent stress and facilitate a calm and relaxed condition among animals. Yet, current mono-anesthesia has been shown to impair neural transmission and hemodynamic integrity. By linking the current state of murine electrophysiology, Ca(2+) imaging and fMRI of anesthetic effects to findings from human studies, this systematic review proposes general principles to design, apply and monitor anesthetic protocols in a more sophisticated way. The further development of balanced multimodal anesthesia, combining two or more drugs with complementary modes of action helps to shape and maintain specific brain states and relevant aspects of murine physiology. Functional connectivity and its dynamic repertoire as assessed by fMRI can be used to make inferences about cortical states and provide additional information about whole-brain functional dynamics. Based on this, a simple and comprehensive functional neurosignature pattern can be determined for use in defining brain states and anesthetic depth in rest and in response to stimuli. Such a signature can be evaluated and shared between labs to indicate the brain state of a mouse during experiments, an important step toward translating findings across species

Functional and structural substrates of increased dosage of Grik4 gene elucidated using multi-modal MRI

Grik4 is the gene responsible for encoding the high-affinity GluK4 subunit of the kainate receptors. Increased dosage of this subunit in the forebrain was linked to an increased level of anxiety, lack of social communication, and depression. On the synaptic level, abnormal synaptic transmission was also reported. The manifestations of this abnormal expression have not been investigated at the circuit level, nor the correlations between those circuits and the abnormal patterns of the behavior previously reported. In this line of work, we aspired to use different non-invasive magnetic resonance imaging (MRI) modalities to elucidate any disturbance that might stem from the increased dosage of Grik4 and how those changes might explain the abnormal behaviors. MRI offers a noninvasive way to look into the intact brain in vivo. Resting-state functional MRI casts light on how the brain function at rest on the network level and has the capability to detect any anomalies that might occur within or between those networks. On the microstructural level, the diffusion MRI is concerned with the underlying features of the tissues, using the diffusion of water molecules as a proxy for that end. Moving more macroscopically, using structural scans, voxel-based morphometry can detect subtle differences in the morphology of the different brain structures. We recorded videos of our animals performing two tasks that have long been linked to anxiety, the open field and the plus-maze tests before acquiring structural and functional scans. Lastly, we recorded blood-oxygenationlevel dependent (BOLD) signals in a different set of animals during electrical stimulation of specific white matter tracts in order to investigate how neuronal activity propagates. Our analysis showed a vast spectrum of changes in the transgenic group relative to the animals in the control group. On the resting-state networks level, we observed an increase in the within-network strength spanning different structures such as the hippocampus, some regions of the cortex, and the hypothalamus. The increased internal coherence or strength in the networks contrasted with a significant reduction in between-networks connectivity for some regions such as parts of the cortex and the hypothalamus, suggesting long-range network decorrelation. Supporting this idea, major white matter (WM) tracts, such as the corpus callosum and the hippocampal commissure, suffered from substantial changes compatible with an important reduction in myelination and/or a decrease in the mean axonal diameter. Macrostructurally speaking, the overexpression of GluK4 subunit had a bimodal effect, with expansion in some cortical areas in the transgenic animals accompanied by a shrinkage in the subcortical regions. Upon stimulating the brain with an electrical current, we noticed a difference in activity propagation between the two hemispheres. In transgenic animals, the evoked activity remained more confined to the stimulated hemisphere, again consistent with an impaired long-range connectivity. The structural changes both, at the micro and macro level, were in tight correlation with different aspects of the behavior including markers of anxiety such as the time spent in the open arms vs the closed arms in the plus-maze test and the time spent in the center vs the corners in the open field test. Our findings reveal how the disruption of kainate receptors, or more globally the glutamate receptors, and the abnormal synaptic transmission can translate into brain-wide changes in connectivity and alter the functional equilibrium between macro-and mesoscopic networks. The postsynaptic enhancement previously reported in the transgenic animals was here reflected in the BOLD signal and measured as an increase in the within-network strength. Importantly, the correlations between the structural changes and the behavior help to put the developmental changes and their behavioral ramifications into context. RESUMEN Grik4 es el gen responsable de codificar la subunidad GluK4 de alta afinidad de los receptores de kainato. El aumento de la dosis de esta subunidad en el prosencéfalo se relacionó con un mayor nivel de ansiedad, falta de comunicación social y depresión. A nivel sináptico, también se informó una transmisión sináptica anormal. Las manifestaciones de esta expresión anormal no se han investigado a nivel de circuito, ni las correlaciones entre esos circuitos y los patrones anormales de la conducta previamente informada. En esta línea de trabajo, aspiramos a utilizar diferentes modalidades de imágenes por resonancia magnética (MRI) no invasivas para dilucidar cualquier alteración que pudiera derivarse del aumento de la dosis de Grik4 y cómo esos cambios podrían explicar los comportamientos anormales. La resonancia magnética ofrece una forma no invasiva de observar el cerebro intacto in vivo. La resonancia magnética funcional en estado de reposo arroja luz sobre cómo funciona el cerebro en reposo en el nivel de la red y tiene la capacidad de detectar cualquier anomalía que pueda ocurrir dentro o entre esas redes. En el nivel microestructural, la resonancia magnética de difusión se ocupa de las características subyacentes de los tejidos utilizando la difusión de moléculas de agua como un proxy para ese fin. Moviéndose más macroscópicamente, utilizando escaneos estructurales, la morfometría basada en vóxeles puede detectar diferencias sutiles en la morfología de las diferentes estructuras cerebrales. Grabamos videos de nuestros animales realizando dos tareas que durante mucho tiempo se han relacionado con la ansiedad, el campo abierto y las pruebas de laberinto positivo antes de adquirir escaneos estructurales y funcionales. Por último, registramos señales dependientes del nivel de oxigenación de la sangre (BOLD) en un grupo diferente de animales durante la estimulación eléctrica de tractos específicos de materia blanca para investigar cómo se propaga la actividad neuronal. Nuestro análisis mostró un amplio espectro de cambios en el grupo transgénico en relación con los animales en el grupo de control. En el nivel de las redes de estado de reposo, observamos un aumento en la fuerza dentro de la red que abarca diferentes estructuras como el hipocampo, algunas regiones de la corteza y el hipotálamo. La mayor coherencia interna o fuerza en las redes contrastó con una reducción significativa en la conectividad entre redes para algunas regiones como partes de la corteza y el hipotálamo, lo que sugiere una descorrelación de redes de largo alcance. Apoyando esta idea, los grandes tractos de materia blanca (WM), como el cuerpo calloso y la comisura del hipocampo, sufrieron cambios sustanciales compatibles con una importante reducción de la mielinización y / o una disminución del diámetro axonal medio. Macroestructuralmente hablando, la sobreexpresión de la subunidad GluK4 tuvo un efecto bimodal, con expansión en algunas áreas corticales en los animales transgénicos acompañada de una contracción en las regiones subcorticales. Al estimular el cerebro con una corriente eléctrica, notamos una diferencia en la propagación de la actividad entre las dos hemiesferas. En los animales transgénicos, la actividad evocada permaneció más confinada al hemisferio estimulado, de nuevo consistente con una conectividad de largo alcance deteriorada. Los cambios estructurales, tanto a nivel micro como macro, estaban en estrecha correlación con diferentes aspectos de la conducta, incluidos marcadores de ansiedad como el tiempo pasado con los brazos abiertos frente a los brazos cerrados en la prueba del laberinto positivo y el tiempo pasado en el centro vs las esquinas en la prueba de campo abierto. Nuestros hallazgos revelan cómo la interrupción de los receptores de kainato, o más globalmente los receptores de glutamato, y la transmisión sináptica anormal pueden traducirse en cambios de conectividad en todo el cerebro y alterar el equilibrio funcional entre las redes macro y mesoscópicas. La mejora postsináptica informada anteriormente en los animales transgénicos se reflejó aquí en la señal BOLD y se midió como un aumento en la fuerza dentro de la red. Es importante destacar que las correlaciones entre los cambios estructurales y elcomportamiento ayudan a contextualizar los cambios en el desarrollo y sus ramificaciones conductuales