Similarity and characterization of structural and functional neural connections within species under isoflurane anesthesia in the common marmoset.

The common marmoset is an essential model for understanding social cognition and neurodegenerative diseases. This study explored the structural and functional brain connectivity in a marmoset under isoflurane anesthesia, aiming to statistically overcome the effects of high inter-individual variability and noise-related confounds such as physiological noise, ensuring robust and reliable data. Similarities and differences in individual subject data, including assessments of functional and structural brain connectivities derived from resting-state functional MRI and diffusion tensor imaging were meticulously captured. The findings highlighted the high consistency of structural neural connections within the species, indicating a stable neural architecture, while functional connectivity under anesthesia displayed considerable variability. Through independent component and dual regression analyses, several distinct brain connectivities were identified, elucidating their characteristics under anesthesia. Insights into the structural and functional features of the marmoset brain from this study affirm its value as a neuroscience research model, promising advancements in the field through fundamental and translational studies.

A whole-brain analysis of functional connectivity and immediate early gene expression reveals functional network shifts after operant learning.

Previous studies of operant learning have addressed neuronal activities and network changes in specific brain areas, such as the striatum, sensorimotor cortex, prefrontal/orbitofrontal cortices, and hippocampus. However, how changes in the whole-brain network are caused by cellular-level changes remains unclear. We, therefore, combined resting-state functional magnetic resonance imaging (rsfMRI) and whole-brain immunohistochemical analysis of early growth response 1 (EGR1), a marker of neural plasticity, to elucidate the temporal and spatial changes in functional networks and underlying cellular processes during operant learning. We used an 11.7-Tesla MRI scanner and whole-brain immunohistochemical analysis of EGR1 in mice during the early and late stages of operant learning. In the operant training, mice received a reward when they pressed left and right buttons alternately, and were punished with a bright light when they made a mistake. A group of mice (n = 22) underwent the first rsfMRI acquisition before behavioral sessions, the second acquisition after 3 training-session-days (early stage), and the third after 21 training-session-days (late stage). Another group of mice (n = 40) was subjected to histological analysis 15 min after the early or late stages of behavioral sessions. Functional connectivity increased between the limbic areas and thalamus or auditory cortex after the early stage of training, and between the motor cortex, sensory cortex, and striatum after the late stage of training. The density of EGR1-immunopositive cells in the motor and sensory cortices increased in both the early and late stages of training, whereas the density in the amygdala increased only in the early stage of training. The subcortical networks centered around the limbic areas that emerged in the early stage have been implicated in rewards, pleasures, and fears. The connectivities between the motor cortex, somatosensory cortex, and striatum that consolidated in the late stage have been implicated in motor learning. Our multimodal longitudinal study successfully revealed temporal shifts in brain regions involved in behavioral learning together with the underlying cellular-level plasticity between these regions. Our study represents a first step towards establishing a new experimental paradigm that combines rsfMRI and immunohistochemistry to link macroscopic and microscopic mechanisms involved in learning.

Increased functional connectivity following ingestion of dried bonito soup.

Soup, including dried bonito broth, is customarily consumed as an umami taste during meals in Japan. Previous functional magnetic resonance imaging (fMRI) studies have investigated neuronal activation following human exposure to carbohydrates and umami substances. However, neuronal activity following ingestion of dried bonito soup has not been investigated. Additionally, recent progress in fMRI has enabled us to investigate the functional connectivity between two anatomically separated regions, such as the default mode network. In this study, we first investigated the altered functional connectivity after ingesting dried bonito soup in healthy volunteers. Functional connectivity in several brain regions, including the connection between the vermis, part of the cerebellum, and bilateral central opercular cortex, was markedly increased after ingesting dried bonito soup, compared to the ingestion of hot water. Physiological scaling showed that satiety was substantially increased by ingesting hot water rather than dried bonito soup. These results indicate that increased functional connectivity reflects the post-ingestive information pathway of dried bonito soup.

Comparison of local activation, functional connectivity, and structural connectivity in the N-back task.

The N-back task is widely used to investigate working memory. Previous functional magnetic resonance imaging (fMRI) studies have shown that local brain activation depends on the difficulty of the N-back task. Recently, changes in functional connectivity and local activation during a task, such as a single-hand movement task, have been reported to give the distinct information. However, previous studies have not investigated functional connectivity changes in the entire brain during N-back tasks. In this study, we compared alterations in functional connectivity and local activation related to the difficulty of the N-back task. Because structural connectivity has been reported to be associated with local activation, we also investigated the relationship between structural connectivity and accuracy in a N-back task using diffusion tensor imaging (DTI). Changes in functional connectivity depend on the difficulty of the N-back task in a manner different from local activation, and the 2-back task is the best method for investigating working memory. This indicates that local activation and functional connectivity reflect different neuronal events during the N-back task. The top 10 structural connectivities associated with accuracy in the 2-back task were locally activated during the 2-back task. Therefore, structural connectivity as well as fMRI will be useful for predicting the accuracy of the 2-back task.

Increased interhemispheric functional connectivity during non-dominant hand movement in right-handed subjects.

Hand preference is one of the behavioral expressions of lateralization in the brain. Previous fMRI studies showed the activation in several regions including the motor cortex and the cerebellum during single-hand movement. However, functional connectivity related to hand preference has not been investigated. Here, we used the generalized psychophysiological interaction (gPPI) approach to investigate the alteration of functional connectivity during single-hand movement from the resting state in right-hand subjects. The functional connectivity in interhemispheric motor-related regions including the supplementary motor area, the precentral gyrus, and the cerebellum was significantly increased during non-dominant hand movement, while functional connectivity was not increased during dominant hand movement. The general linear model (GLM) showed activation in contralateral supplementary motor area, contralateral precentral gyrus, and ipsilateral cerebellum during right- or left-hand movement. These results indicate that a combination of GLM and gPPI analysis can detect the lateralization of hand preference more clearly.

Functional ultrasound reveals effects of MRI acoustic noise on brain function.

Loud acoustic noise from the scanner during functional magnetic resonance imaging (fMRI) can affect functional connectivity (FC) observed in the resting state, but the exact effect of the MRI acoustic noise on resting state FC is not well understood. Functional ultrasound (fUS) is a neuroimaging method that visualizes brain activity based on relative cerebral blood volume (rCBV), a similar neurovascular coupling response to that measured by fMRI, but without the audible acoustic noise. In this study, we investigated the effects of different acoustic noise levels (silent, 80 dB, and 110 dB) on FC by measuring resting state fUS (rsfUS) in awake mice in an environment similar to fMRI measurement. Then, we compared the results to those of resting state fMRI (rsfMRI) conducted using an 11.7 Tesla scanner. RsfUS experiments revealed a significant reduction in FC between the retrosplenial dysgranular and auditory cortexes (0.56 ± 0.07 at silence vs 0.05 ± 0.05 at 110 dB, p=.01) and a significant increase in FC anticorrelation between the infralimbic and motor cortexes (-0.21 ± 0.08 at silence vs -0.47 ± 0.04 at 110 dB, p=.017) as acoustic noise increased from silence to 80 dB and 110 dB, with increased consistency of FC patterns between rsfUS and rsfMRI being found with the louder noise conditions. Event-related auditory stimulation experiments using fUS showed strong positive rCBV changes (16.5% ± 2.9% at 110 dB) in the auditory cortex, and negative rCBV changes (-6.7% ± 0.8% at 110 dB) in the motor cortex, both being constituents of the brain network that was altered by the presence of acoustic noise in the resting state experiments. Anticorrelation between constituent brain regions of the default mode network (such as the infralimbic cortex) and those of task-positive sensorimotor networks (such as the motor cortex) is known to be an important feature of brain network antagonism, and has been studied as a biological marker of brain disfunction and disease. This study suggests that attention should be paid to the acoustic noise level when using rsfMRI to evaluate the anticorrelation between the default mode network and task-positive sensorimotor network.

Brain-wide mapping of resting-state networks in mice using high-frame rate functional ultrasound.

Functional ultrasound (fUS) imaging is a method for visualizing deep brain activity based on cerebral blood volume changes coupled with neural activity, while functional MRI (fMRI) relies on the blood-oxygenation-level-dependent signal coupled with neural activity. Low-frequency fluctuations (LFF) of fMRI signals during resting-state can be measured by resting-state fMRI (rsfMRI), which allows functional imaging of the whole brain, and the distributions of resting-state network (RSN) can then be estimated from these fluctuations using independent component analysis (ICA). This procedure provides an important method for studying cognitive and psychophysiological diseases affecting specific brain networks. The distributions of RSNs in the brain-wide area has been reported primarily by rsfMRI. RSNs using rsfMRI are generally computed from the time-course of fMRI signals for more than 5 min. However, a recent dynamic functional connectivity study revealed that RSNs are still not perfectly stable even after 10 min. Importantly, fUS has a higher temporal resolution and stronger correlation with neural activity compared with fMRI. Therefore, we hypothesized that fUS applied during the resting-state for a shorter than 5 min would provide similar RSNs compared to fMRI. High temporal resolution rsfUS data were acquired at 10 Hz in awake mice. The quality of the default mode network (DMN), a well-known RSN, was evaluated using signal-noise separation (SNS) applied to different measurement durations of rsfUS. The results showed that the SNS did not change when the measurement duration was increased to more than 210 s. Next, we measured short-duration rsfUS multi-slice measurements in the brain-wide area. The results showed that rsfUS with the short duration succeeded in detecting RSNs distributed in the brain-wide area consistent with RSNs detected by 11.7-T MRI under awake conditions (medial prefrontal cortex and cingulate cortex in the anterior DMN, retrosplenial cortex and visual cortex in the posterior DMN, somatosensory and motor cortexes in the lateral cortical network, thalamus, dorsal hippocampus, and medial cerebellum), confirming the reliability of the RSNs detected by rsfUS. However, bilateral RSNs located in the secondary somatosensory cortex, ventral hippocampus, auditory cortex, and lateral cerebellum extracted from rsfUS were different from the unilateral RSNs extracted from rsfMRI. These findings indicate the potential of rsfUS as a method for analyzing functional brain networks and should encourage future research to elucidate functional brain networks and their relationships with disease model mice.

Micro-magnetic resonance imaging of ex vivo mouse embryos with potato starch suspension.

Potato starch suspension (PSS) holds promise as a solution to issues, such as air bubbles and specimen motion, associated with micro-magnetic resonance imaging (micro-MRI) of ex vivo embryos. Here, we present a protocol for using PSS when scanning specimens with micro-MRI. We describe steps for preparing samples and potato starch with phosphate-buffered saline. We then detail steps for specimen immersion and micro-MRI scanning. This protocol will enable micro-MRI of not only embryos but also other specimens, such as insects. For complete details on the use and execution of this protocol, please refer to Tsurugizawa et al.1.

Inflammation and subsequent nociceptor sensitization in the bone marrow are involved in an animal model of osteoarthritis pain.

AIMS: This study aimed to determine whether pathological changes in the bone marrow cause Osteoarthritis (OA) pain based on magnetic resonance imaging (MRI), immunohistochemistry, and electrophysiology. MAIN METHODS: Adjuvant-induced arthritis (AIA) was achieved by injecting 150 µL of complete Freund's adjuvant into the right knee joints of male Sprague-Dawley rats. AIA rats were compared with saline-injected rats. KEY FINDINGS: AIA significantly induced mechanical hyperalgesia and spontaneous pain in the right hind paw 1-14 days after induction. Intratibial injection of 50 µL of 1 % lidocaine significantly suppressed AIA-induced mechanical hyperalgesia (p = 0.0001) and spontaneous pain (p = 0.0006) 3 days after induction. In T2-weighted MRI, AIA induced high-signal intensity within the proximal tibial metaphysis, and the mean T2 values in this area significantly increased on days 3 (p = 0.0043) and 14 (p = 0.0012) after induction. AIA induced intraosseous edema and significantly increased the number of intraosseous granulocytes on days 3 (p < 0.0001) and 14 (p < 0.0001) after induction. The electrophysiological study on days 3-7 after induction showed significantly increased spontaneous firing rates (p = 0.0166) and evoked responses to cutaneous stimuli (brush, p < 0.0001; pinching, p = 0.0359) in the right hind paw plantar surface and intratibial stimuli (p = 0.0002) in wide-dynamic-range neurons of the spinal dorsal horn. SIGNIFICANCE: Intraosseous changes caused by OA induce hypersensitivity in the sensory afferents innervating bone marrow may be involved in OA pain. Novel bone marrow-targeted therapies could be beneficial for treating OA pain.

Utilization of potato starch suspension for MR-microimaging in ex vivo mouse embryos.

Magnetic resonance (MR) microimaging of the mouse embryo is a promising tool to noninvasively investigate the microstructure of the brain of a developing mouse. The proton-free fluid is used for the liquid surrounding the specimen in MR microimaging, but the potential issue of image quality remains due to the air bubbles on the specimen and the retained water proton in the curvature of the embryo. Furthermore, the specimen may move during the scanning, resulting in motion artifact. Here, we developed the new concept of the ex vivo microimaging protocol with the robust method using the potato starch-containing biological polymers. Potato starch suspension with PBS significantly reduced T1 and T2 signal intensity of the suspension and strongly suppressed the motion of the embryo. Furthermore, potato starch-PBS suspension is stable for long-time scanning at room temperature. These results indicate the utility of potato starch suspension for MR microimaging in mouse embryos.

Translational Magnetic Resonance Imaging in Autism Spectrum Disorder From the Mouse Model to Human.

Autism spectrum disorder (ASD) is a heterogeneous syndrome characterized by behavioral features such as impaired social communication, repetitive behavior patterns, and a lack of interest in novel objects. A multimodal neuroimaging using magnetic resonance imaging (MRI) in patients with ASD shows highly heterogeneous abnormalities in function and structure in the brain associated with specific behavioral features. To elucidate the mechanism of ASD, several ASD mouse models have been generated, by focusing on some of the ASD risk genes. A specific behavioral feature of an ASD mouse model is caused by an altered gene expression or a modification of a gene product. Using these mouse models, a high field preclinical MRI enables us to non-invasively investigate the neuronal mechanism of the altered brain function associated with the behavior and ASD risk genes. Thus, MRI is a promising translational approach to bridge the gap between mice and humans. This review presents the evidence for multimodal MRI, including functional MRI (fMRI), diffusion tensor imaging (DTI), and volumetric analysis, in ASD mouse models and in patients with ASD and discusses the future directions for the translational study of ASD.

Potential of Multiscale Astrocyte Imaging for Revealing Mechanisms Underlying Neurodevelopmental Disorders.

Astrocytes provide trophic and metabolic support to neurons and modulate circuit formation during development. In addition, astrocytes help maintain neuronal homeostasis through neurovascular coupling, blood-brain barrier maintenance, clearance of metabolites and nonfunctional proteins via the glymphatic system, extracellular potassium buffering, and regulation of synaptic activity. Thus, astrocyte dysfunction may contribute to a myriad of neurological disorders. Indeed, astrocyte dysfunction during development has been implicated in Rett disease, Alexander's disease, epilepsy, and autism, among other disorders. Numerous disease model mice have been established to investigate these diseases, but important preclinical findings on etiology and pathophysiology have not translated into clinical interventions. A multidisciplinary approach is required to elucidate the mechanism of these diseases because astrocyte dysfunction can result in altered neuronal connectivity, morphology, and activity. Recent progress in neuroimaging techniques has enabled noninvasive investigations of brain structure and function at multiple spatiotemporal scales, and these technologies are expected to facilitate the translation of preclinical findings to clinical studies and ultimately to clinical trials. Here, we review recent progress on astrocyte contributions to neurodevelopmental and neuropsychiatric disorders revealed using novel imaging techniques, from microscopy scale to mesoscopic scale.

Impact of anesthesia on static and dynamic functional connectivity in mice.

A few studies have compared the static functional connectivity between awake and lightly anesthetized states in rodents by resting-state fMRI. However, impact of light anesthesia on static and dynamic fluctuations in functional connectivity has not been fully understood. Here, we developed a resting-state fMRI protocol to perform awake and anesthetized functional MRI in the same mice. Static functional connectivity showed a widespread decrease under light anesthesia, such as when under isoflurane or a mixture of isoflurane and medetomidine. Several interhemispheric and subcortical connections were key connections for anesthetized condition from awake state. Dynamic functional connectivity demonstrates the shift from frequent broad connections across the cortex, the hypothalamus, and the auditory-visual cortex to frequent local connections within the cortex only under light anesthesia compared with awake state. Fractional amplitude of low frequency fluctuation in the thalamic nuclei decreased under both anesthesia. These results indicate that typical anesthetics for functional MRI alters the spatiotemporal profile of the dynamic brain network in subcortical regions, including the thalamic nuclei and limbic system.

Fasting prevents medetomidine-induced hyperglycaemia and alterations of neurovascular coupling in the somatosensory cortex of the rat during noxious stimulation.

Medetomidine and isoflurane are commonly used for general anaesthesia in fMRI studies, but they alter cerebral blood flow (CBF) regulation and neurovascular coupling (NVC). In addition, medetomidine induces hypoinsulinemia and hyperglycaemia, which also alter CBF regulation and NVC. Furthermore, sudden changes in arterial pressure induced by noxious stimulation may affect NVC differently under medetomidine and isoflurane anaesthesia, considering their different effects on vascular functions. The first objective of this study was to compare NVC under medetomidine and isoflurane anaesthesia during noxious stimulation. The second objective was to examine whether fasting may improve NVC by reducing medetomidine-induced hyperglycaemia. In male Wister rats, noxious electrical stimulation was applied to the sciatic nerve in fasted or non-fasted animals. CBF and local field potentials (LFP) were recorded in the somatosensory cortex to assess NVC (CBF/LFP ratio). The CBF/LFP ratio was increased by medetomidine compared with isoflurane (p = 0.004), but this effect was abolished by fasting (p = 0.8). Accordingly, medetomidine produced a threefold increase in blood glucose (p < 0.001), but this effect was also abolished by fasting (p = 0.3). This indicates that isoflurane and medetomidine anaesthesia alter NVC differently, but the undesirable glucose dependent effects of medetomidine on NVC can be prevented by fasting.

Cranioplastic Surgery and Acclimation Training for Awake Mouse fMRI.

MRI is a promising tool for translational research to link brain function and structure in animal models of disease to patients with neuropsychiatric disorders. However, given that mouse functional MRI (fMRI) typically relies on anesthetics to suppress head motion and physiological noise, it has been difficult to directly compare brain fMRI in anesthetized mice with that in conscious patients. Here, we developed a new system to acquire fMRI in awake mice, which includes a head positioner and dedicated radio frequency coil. The system was used to investigate functional brain networks in conscious mice, with the goal of enabling future studies to bridge fMRI of disease model animals with human fMRI. Cranioplastic surgery was performed to affix the head mount and the cupped-hand handling method was performed to minimize stress during MRI scanning. Here we describe the new mouse fMRI system, cranioplastic surgery and acclimation protocol. Graphic abstract: Awake fMRI system to investigate the neuronal activity in awaked mice.

fMRI detects bilateral brain network activation following unilateral chemogenetic activation of direct striatal projection neurons.

Abnormal structural and functional connectivity in the striatum during neurological disorders has been reported using functional magnetic resonance imaging (fMRI), although the effects of cell-type specific neuronal stimulation on fMRI and related behavioral alterations are not well understood. In this study, we combined DREADD technology with fMRI ("chemo-fMRI") to investigate alterations of spontaneous neuronal activity. These were induced by the unilateral activation of dopamine D1 receptor-expressing neurons (D1-neurons) in the mouse dorsal striatum (DS). After clozapine (CLZ) stimulation of the excitatory DREADD expressed in D1-neurons, the fractional amplitude of low frequency fluctuations (fALFF) increased bilaterally in the medial thalamus, nucleus accumbens and cortex. In addition, we found that the gamma-band of local field potentials was increased in the stimulated DS and cortex bilaterally. These results provide insights for better interpretation of cell type-specific activity changes in fMRI.

Diffusion MRI reveals in vivo and non-invasively changes in astrocyte function induced by an aquaporin-4 inhibitor.

The Glymphatic System (GS) has been proposed as a mechanism to clear brain tissue from waste. Its dysfunction might lead to several brain pathologies, including the Alzheimer's disease. A key component of the GS and brain tissue water circulation is the astrocyte which is regulated by acquaporin-4 (AQP4), a membrane-bound water channel on the astrocytic end-feet. Here we investigated the potential of diffusion MRI to monitor astrocyte activity in a mouse brain model through the inhibition of AQP4 channels with TGN-020. Upon TGN-020 injection, we observed a significant decrease in the Sindex, a diffusion marker of tissue microstructure, and a significant increase of the water diffusion coefficient (sADC) in cerebral cortex and hippocampus compared to saline injection. These results indicate the suitability of diffusion MRI to monitor astrocytic activity in vivo and non-invasively.

Differential effects of aquaporin-4 channel inhibition on BOLD fMRI and diffusion fMRI responses in mouse visual cortex.

The contribution of astrocytes to the BOLD fMRI and DfMRI responses in visual cortex of mice following visual stimulation was investigated using TGN-020, an aquaporin 4 (AQP4) channel blocker, acting as an astrocyte function perturbator. Under TGN-020 injection the amplitude of the BOLD fMRI response became significantly higher. In contrast no significant changes in the DfMRI responses and the electrophysiological responses were observed. Those results further confirm the implications of astrocytes in the neurovascular coupling mechanism underlying BOLD fMRI, but not in the DfMRI responses which remained unsensitive to astrocyte function perturbation.

Author Correction: The impact of fasting on resting state brain networks in mice.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Awake functional MRI detects neural circuit dysfunction in a mouse model of autism.

MRI has potential as a translational approach from rodents to humans. However, given that mouse functional MRI (fMRI) uses anesthetics for suppression of motion, it has been difficult to directly compare the result of fMRI in "unconsciousness" disease model mice with that in "consciousness" patients. We develop awake fMRI to investigate brain function in 15q dup mice, a copy number variation model of autism. Compared to wild-type mice, we find that 15q dup is associated with whole-brain functional hypoconnectivity and diminished fMRI responses to odors of stranger mice. Ex vivo diffusion MRI reveals widespread anomalies in white matter ultrastructure in 15q dup mice, suggesting a putative anatomical substrate for these functional hypoconnectivity. We show that d-cycloserine (DCS) treatment partially normalizes these anormalies in the frontal cortex of 15q dup mice and rescues some social behaviors. Our results demonstrate the utility of awake rodent fMRI and provide a rationale for further investigation of DCS therapy.