Anterior cingulate cortex-related functional hyperconnectivity underlies sensory hypersensitivity in Grin2b-mutant mice.

Sensory abnormalities are observed in ~90% of individuals with autism spectrum disorders (ASD), but the underlying mechanisms are poorly understood. GluN2B, an NMDA receptor subunit that regulates long-term depression and circuit refinement during brain development, has been strongly implicated in ASD, but whether GRIN2B mutations lead to sensory abnormalities remains unclear. Here, we report that Grin2b-mutant mice show behavioral sensory hypersensitivity and brain hyperconnectivity associated with the anterior cingulate cortex (ACC). Grin2b-mutant mice with a patient-derived C456Y mutation (Grin2bC456Y/+) show sensory hypersensitivity to mechanical, thermal, and electrical stimuli through supraspinal mechanisms. c-fos and functional magnetic resonance imaging indicate that the ACC is hyperactive and hyperconnected with other brain regions under baseline and stimulation conditions. ACC pyramidal neurons show increased excitatory synaptic transmission. Chemogenetic inhibition of ACC pyramidal neurons normalizes ACC hyperconnectivity and sensory hypersensitivity. These results suggest that GluN2B critically regulates ASD-related cortical connectivity and sensory brain functions.

Loss of Katnal2 leads to ependymal ciliary hyperfunction and autism-related phenotypes in mice.

Autism spectrum disorders (ASD) frequently accompany macrocephaly, which often involves hydrocephalic enlargement of brain ventricles. Katnal2 is a microtubule-regulatory protein strongly linked to ASD, but it remains unclear whether Katnal2 knockout (KO) in mice leads to microtubule- and ASD-related molecular, synaptic, brain, and behavioral phenotypes. We found that Katnal2-KO mice display ASD-like social communication deficits and age-dependent progressive ventricular enlargements. The latter involves increased length and beating frequency of motile cilia on ependymal cells lining ventricles. Katnal2-KO hippocampal neurons surrounded by enlarged lateral ventricles show progressive synaptic deficits that correlate with ASD-like transcriptomic changes involving synaptic gene down-regulation. Importantly, early postnatal Katnal2 re-expression prevents ciliary, ventricular, and behavioral phenotypes in Katnal2-KO adults, suggesting a causal relationship and a potential treatment. Therefore, Katnal2 negatively regulates ependymal ciliary function and its deletion in mice leads to ependymal ciliary hyperfunction and hydrocephalus accompanying ASD-related behavioral, synaptic, and transcriptomic changes.

Separation of bimodal fMRI responses in mouse somatosensory areas into V1 and non-V1 contributions.

Multisensory integration is necessary for the animal to survive in the real world. While conventional methods have been extensively used to investigate the multisensory integration process in various brain areas, its long-range interactions remain less explored. In this study, our goal was to investigate interactions between visual and somatosensory networks on a whole-brain scale using 15.2-T BOLD fMRI. We compared unimodal to bimodal BOLD fMRI responses and dissected potential cross-modal pathways with silencing of primary visual cortex (V1) by optogenetic stimulation of local GABAergic neurons. Our data showed that the influence of visual stimulus on whisker activity is higher than the influence of whisker stimulus on visual activity. Optogenetic silencing of V1 revealed that visual information is conveyed to whisker processing via both V1 and non-V1 pathways. The first-order ventral posteromedial thalamic nucleus (VPM) was functionally affected by non-V1 sources, while the higher-order posterior medial thalamic nucleus (POm) was predominantly modulated by V1 but not non-V1 inputs. The primary somatosensory barrel field (S1BF) was influenced by both V1 and non-V1 inputs. These observations provide valuable insights for into the integration of whisker and visual sensory information.

No replication of direct neuronal activity-related (DIANA) fMRI in anesthetized mice.

Direct imaging of neuronal activity (DIANA) by functional magnetic resonance imaging (fMRI) could be a revolutionary approach for advancing systems neuroscience research. To independently replicate this observation, we performed fMRI experiments in anesthetized mice. The blood oxygenation level-dependent (BOLD) response to whisker stimulation was reliably detected in the primary barrel cortex before and after DIANA experiments; however, no DIANA-like fMRI peak was observed in individual animals' data with the 50 to 300 trials. Extensively averaged data involving 1050 trials in six mice showed a flat baseline and no detectable neuronal activity-like fMRI peak. However, spurious, nonreplicable peaks were found when using a small number of trials, and artifactual peaks were detected when some outlier-like trials were excluded. Further, no detectable DIANA peak was observed in the BOLD-responding thalamus from the selected trials with the neuronal activity-like reference function in the barrel cortex. Thus, we were unable to replicate the previously reported results without data preselection.

Mapping cerebral perfusion in mice under various anesthesia levels using highly sensitive BOLD MRI with transient hypoxia.

Cerebral perfusion is critical for the early detection of neurological diseases and for effectively monitoring disease progression and treatment responses. Mouse models are widely used in brain research, often under anesthesia, which can affect vascular physiology. However, the impact of anesthesia on regional cerebral blood volume and flow in mice has not been thoroughly investigated. In this study, we have developed a whole-brain perfusion MRI approach by using a 5-second nitrogen gas stimulus under inhalational anesthetics to induce transient BOLD dynamic susceptibility contrast (DSC). This method proved to be highly sensitive, repeatable within each imaging session, and across four weekly sessions. Relative cerebral blood volumes measured by BOLD DSC agree well with those by contrast agents. Quantitative cerebral blood volume and flow metrics were successfully measured in mice under dexmedetomidine and various isoflurane doses using both total vasculature-sensitive gradient-echo and microvasculature-sensitive spin-echo BOLD MRI. Dexmedetomidine reduces cerebral perfusion, while isoflurane increases cerebral perfusion in a dose-dependent manner.

Sticky and Strain-Gradient Artificial Epineurium for Sutureless Nerve Repair in Rodents and Nonhuman Primates.

The need for the development of soft materials capable of stably adhering to nerve tissues without any suturing followed by additional damages is at the fore at a time when success in postoperative recovery depends largely on the surgical experience and/or specialized microsuturing skills of the surgeon. Despite fully recognizing such prerequisite conditions, designing the materials with robust adhesion to wet nerves as well as acute/chronic anti-inflammation remains to be resolved. Herein, a sticky and strain-gradient artificial epineurium (SSGAE) that overcomes the most critically challenging aspect for realizing sutureless repair of severely injured nerves is presented. In this regard, the SSGAE with a skin-inspired hierarchical structure entailing strain-gradient layers, anisotropic Janus layers including hydrophobic top and hydrophilic bottom surfaces, and synergistic self-healing capabilities enables immediate and stable neurorrhaphy in both rodent and nonhuman primate models, indicating that the bioinspired materials strategy significantly contributes to translational medicine for effective peripheral nerve repair.

Interaction Between a High-Fat Diet and Tau Pathology in Mice: Implications for Alzheimer's Disease.

BACKGROUND: Obesity is a modifiable risk factor for Alzheimer's disease (AD). However, its relation with tau pathology (i.e., aberrant tau protein behavior in tauopathies such as AD) has been inconclusive. OBJECTIVE: This study investigated the interaction between a high-fat diet (HFD) and tau pathology in adult male mice. METHODS: Transgenic mice overexpressing human P301S Tau (those with the pathology) and wild-type (WT) littermates were subjected to behavioral tests, functional magnetic resonance imaging (fMRI), diffusion tensor imaging (DTI), and western blotting analysis to investigate the effects of prolonged HFD versus regular diet during adulthood. RESULTS: HFD increased body weight in both WT and P301S mice but had minimal effect on blood glucose levels. The brain response to HFD was tau genotype-specific. WT mice exhibited decreased recognition memory and enhanced network connectivity in fMRI, while P301S mice exhibited white matter tract disorganization in DTI as the sole significant finding. The reduction of insulin receptor ß, insulin downstream signaling, neuronal nuclear protein, CD68-positive phagocytic activity, and myelin basic protein level were confined to the cortex of WT mice. In contrast to P301S mice, WT mice showed significant changes in the tau protein and its phosphorylation levels along with increased soluble neurofilament light levels in the hippocampus. CONCLUSIONS: HFD-induced brain dysfunction and pathological changes were blunted in mice with the pathology and more profound in healthy mice. Our findings highlight the need to consider this interaction between obesity and tau pathology when tailoring treatment strategies for AD and other tauopathies.

No Replication of Direct Neuronal Activity-related (DIANA) fMRI in Anesthetized Mice.

Toi et al. (Science, 378, 160-168, 2022) reported direct imaging of neuronal activity (DIANA) by fMRI in anesthetized mice at 9.4 T, which could be a revolutionary approach for advancing systems neuroscience research. There have been no independent replications of this observation to date. We performed fMRI experiments in anesthetized mice at an ultrahigh field of 15.2 T using the identical protocol as in their paper. The BOLD response to whisker stimulation was reliably detected in the primary barrel cortex before and after DIANA experiments; however, no direct neuronal activity-like fMRI peak was observed in individual animals' data with the 50-300 trials used in the DIANA publication. Extensively averaged data involving 1,050 trials in 6 mice (1,050×54 = 56,700 stimulus events) and having a temporal signal-to-noise ratio of 7,370, showed a flat baseline and no detectable neuronal activity-like fMRI peak. Thus we were unable to replicate the previously reported results using the same methods, despite a much higher number of trials, a much higher temporal signal-to-noise ratio, and a much higher magnetic field strength. We were able to demonstrate spurious, non-replicable peaks when using a small number of trials. It was only when performing the inappropriate approach of excluding outliers not conforming to the expected temporal characteristics of the response did we see a clear signal change; however, these signals were not observed when such a outlier elimination approach was not used.

Miniaturized MR-compatible ultrasound system for real-time monitoring of acoustic effects in mice using high-resolution MRI.

Visualization of focused ultrasound in high spatial and temporal resolution is crucial for accurately and precisely targeting brain regions noninvasively. Magnetic resonance imaging (MRI) is the most widely used noninvasive tool for whole-brain imaging. However, focused ultrasound studies employing high-resolution (> 9.4 T) MRI in small animals are limited by the small size of the radiofrequency (RF) volume coil and the noise sensitivity of the image to external systems such as bulky ultrasound transducers. This technical note reports a miniaturized ultrasound transducer system packaged directly above a mouse brain for monitoring ultrasound-induced effects using high-resolution 9.4 T MRI. Our miniaturized system integrates MR-compatible materials with electromagnetic (EM) noise reduction techniques to demonstrate echo-planar imaging (EPI) signal changes in the mouse brain at various ultrasound acoustic intensities. The proposed ultrasound-MRI system will enable extensive research in the expanding field of ultrasound therapeutics.

Theory of chemical exchange saturation transfer MRI in the context of different magnetic fields.

The article from this special issue was previously published in NMR In Biomedicine , Volume 35, Issue 11, 2022. For completeness we are including the title page of the article below. The full text of the article can be read in Issue 35:11 on Wiley Online Library: https://doi.org/10.1002/nbm.4789.

Seizure-induced neutrophil adhesion in brain capillaries leads to a decrease in postictal cerebral blood flow.

Cerebral hypoperfusion has been proposed as a potential cause of postictal neurological dysfunction in epilepsy, but its underlying mechanism is still unclear. We show that a 30% reduction in postictal cerebral blood flow (CBF) has two contributing factors: the early hypoperfusion up to ∼30 min post-seizure was mainly induced by arteriolar constriction, while the hypoperfusion that persisted for over an hour was due to increased capillary stalling induced by neutrophil adhesion to brain capillaries, decreased red blood cell (RBC) flow accompanied by constriction of capillaries and venules, and elevated intercellular adhesion molecule-1 (ICAM-1) expression. Administration of antibodies against the neutrophil marker Ly6G and against LFA-1, which mediates adhesive interactions with ICAM-1, prevented neutrophil adhesion and recovered the prolonged CBF reductions to control levels. Our findings provide evidence that seizure-induced neutrophil adhesion to cerebral microvessels via ICAM-1 leads to prolonged postictal hypoperfusion, which may underlie neurological dysfunction in epilepsy.

Parvalbumin interneuron activity drives fast inhibition-induced vasoconstriction followed by slow substance P-mediated vasodilation.

The role of parvalbumin (PV) interneurons in vascular control is poorly understood. Here, we investigated the hemodynamic responses elicited by optogenetic stimulation of PV interneurons using electrophysiology, functional magnetic resonance imaging (fMRI), wide-field optical imaging (OIS), and pharmacological applications. As a control, forepaw stimulation was used. Stimulation of PV interneurons in the somatosensory cortex evoked a biphasic fMRI response in the photostimulation site and negative fMRI signals in projection regions. Activation of PV neurons engaged two separable neurovascular mechanisms in the stimulation site. First, an early vasoconstrictive response caused by the PV-driven inhibition is sensitive to the brain state affected by anesthesia or wakefulness. Second, a later ultraslow vasodilation lasting a minute is closely dependent on the sum of interneuron multiunit activities, but is not due to increased metabolism, neural or vascular rebound, or increased glial activity. The ultraslow response is mediated by neuropeptide substance P (SP) released from PV neurons under anesthesia, but disappears during wakefulness, suggesting that SP signaling is important for vascular regulation during sleep. Our findings provide a comprehensive perspective about the role of PV neurons in controlling the vascular response.

Whole-brain mapping of effective connectivity by fMRI with cortex-wide patterned optogenetics.

Functional magnetic resonance imaging (fMRI) with optogenetic neural manipulation is a powerful tool that enables brain-wide mapping of effective functional networks. To achieve flexible manipulation of neural excitation throughout the mouse cortex, we incorporated spatiotemporal programmable optogenetic stimuli generated by a digital micromirror device into an MRI scanner via an optical fiber bundle. This approach offered versatility in space and time in planning the photostimulation pattern, combined with in situ optical imaging and cell-type-specific or circuit-specific genetic targeting in individual mice. Brain-wide effective connectivity obtained by fMRI with optogenetic stimulation of atlas-based cortical regions is generally congruent with anatomically defined axonal tracing data but is affected by the types of anesthetics that act selectively on specific connections. fMRI combined with flexible optogenetics opens a new path to investigate dynamic changes in functional brain states in the same animal through high-throughput brain-wide effective connectivity mapping.

Autodisplay of streptococcal protein G for construction of an orientation-controlled immunoaffinity layer.

An immunoaffinity layer with orientation-controlled antibodies was constructed to express streptococcal protein G in Escherichia coli cells using autodisplay technology. The sequence of protein G, a specific IgG-binding protein, was inserted into the autodisplay vector using recombinant technology and the constructed plasmid vector was transformed into E. coli cells. Protein G was confirmed to be autodisplayed with a high density of 2 × 105 copies per cell by SDS-PAGE analysis, and its IgG-binding affinity was confirmed by fluorescence microscopy. Autodisplayed protein G showed higher affinity than the IgG-binding Z-domain for goat IgG. Immunoassays based on E. coli cells were established to detect horseradish peroxidase (HRP) and C-reactive protein (CRP). Protein G autodisplaying E. coli cells were utilized as a solid support and immunoassays showed improved sensitivity by orientation control of autodisplayed protein G. The outer membrane (OM) of protein G autodisplaying E. coli was isolated and layered to construct an immunoaffinity layer. The OM was coated on a microplate to perform the immunoassays, which showed limits of detection of 5 and 0.2 ng mL-1 for HRP and CRP, respectively. An OM layer with autodisplayed protein G was applied as the immunoaffinity layer of a surface plasmon resonance (SPR) biosensor. After CRP detection, the SPR responses showed good linearity, with an R2 value of 0.99. The immunoaffinity layer with orientation control by autodisplayed protein G was confirmed to be applicable in immunoassays and immunosensors to improve sensitivity.

General-Purpose Ultrasound Neuromodulation System for Chronic, Closed-Loop Preclinical Studies in Freely Behaving Rodents.

Transcranial focused ultrasound stimulation (tFUS) is an effective noninvasive treatment modality for brain disorders with high clinical potential. However, the therapeutic effects of ultrasound neuromodulation are not widely explored due to limitations in preclinical systems. The current preclinical studies are head-fixed, anesthesia-dependent, and acute, limiting clinical translatability. Here, this work reports a general-purpose ultrasound neuromodulation system for chronic, closed-loop preclinical studies in freely behaving rodents. This work uses microelectromechanical systems (MEMS) technology to design and fabricate a small and lightweight transducer capable of artifact-free stimulation and simultaneous neural recording. Using the general-purpose system, it can be observed that state-dependent ultrasound neuromodulation of the prefrontal cortex increases rapid eye movement (REM) sleep and protects spatial working memory to REM sleep deprivation. The system will allow explorative studies in brain disease therapeutics and neuromodulation using ultrasound stimulation for widespread clinical adoption.

Optogenetic fMRI for Brain-Wide Circuit Analysis of Sensory Processing.

Sensory processing is a complex neurological process that receives, integrates, and responds to information from one's own body and environment, which is closely related to survival as well as neurological disorders. Brain-wide networks of sensory processing are difficult to investigate due to their dynamic regulation by multiple brain circuits. Optogenetics, a neuromodulation technique that uses light-sensitive proteins, can be combined with functional magnetic resonance imaging (ofMRI) to measure whole-brain activity. Since ofMRI has increasingly been used for investigating brain circuits underlying sensory processing for over a decade, we systematically reviewed recent ofMRI studies of sensory circuits and discussed the challenges of optogenetic fMRI in rodents.

Improved laminar specificity and sensitivity by combining SE and GE BOLD signals.

The most widely used gradient-echo (GE) blood oxygenation level-dependent (BOLD) contrast has high sensitivity, but low specificity due to draining vein contributions, while spin-echo (SE) BOLD approach at ultra-high magnetic fields is highly specific to neural active sites but has lower sensitivity. To obtain high specificity and sensitivity, we propose to utilize a vessel-size-sensitive filter to the GE-BOLD signal, which suppresses macrovascular contributions and to combine selectively retained microvascular GE-BOLD signals with the SE-BOLD signals. To investigate our proposed idea, fMRI with 0.8 mm isotropic resolution was performed on the primary motor and sensory cortices in humans at 7 T by implementing spin- and gradient-echo (SAGE) echo planar imaging (EPI) acquisition. Microvascular-passed sigmoidal filters were designed based upon the vessel-size-sensitive ΔR2*/ΔR2 value for retaining GE-BOLD signals originating from venous vessels with ≤ 45 µm and ≤ 65 µm diameter. Unlike GE-BOLD fMRI, the laminar profile of SAGE-BOLD fMRI with the vessel-size-sensitive filter peaked at ∼ 1.0 mm from the surface of the primary motor and sensory cortices, demonstrating an improvement of laminar specificity over GE-BOLD fMRI. Also, the functional sensitivity of SAGE BOLD at middle layers (0.75-1.5 mm) was improved by ∼ 80% to ∼100% when compared with SE BOLD. In summary, we showed that combined GE- and SE-BOLD fMRI with the vessel-size-sensitive filter indeed yielded improved laminar specificity and sensitivity and is therefore an excellent tool for high spatial resolution ultra-high filed (UHF)-fMRI studies for resolving mesoscopic functional units.

Postnatal age-differential ASD-like transcriptomic, synaptic, and behavioral deficits in Myt1l-mutant mice.

Myelin transcription factor 1 like (Myt1l), a zinc-finger transcription factor, promotes neuronal differentiation and is implicated in autism spectrum disorder (ASD) and intellectual disability. However, it remains unclear whether Myt1l promotes neuronal differentiation in vivo and its deficiency in mice leads to disease-related phenotypes. Here, we report that Myt1l-heterozygous mutant (Myt1l-HT) mice display postnatal age-differential ASD-related phenotypes: newborn Myt1l-HT mice, with strong Myt1l expression, show ASD-like transcriptomic changes involving decreased synaptic gene expression and prefrontal excitatory synaptic transmission and altered righting reflex. Juvenile Myt1l-HT mice, with markedly decreased Myt1l expression, display reverse ASD-like transcriptomes, increased prefrontal excitatory transmission, and largely normal behaviors. Adult Myt1l-HT mice show ASD-like transcriptomes involving astrocytic and microglial gene upregulation, increased prefrontal inhibitory transmission, and behavioral deficits. Therefore, Myt1l haploinsufficiency leads to ASD-related phenotypes in newborn mice, which are temporarily normalized in juveniles but re-appear in adults, pointing to continuing phenotypic changes long after a marked decrease of Myt1l expression in juveniles.

Whole-brain perfusion mapping in mice by dynamic BOLD MRI with transient hypoxia.

Non-invasive mapping of cerebral perfusion is critical for understanding neurovascular and neurodegenerative diseases. However, perfusion MRI methods cannot be easily implemented for whole-brain studies in mice because of their small size. To overcome this issue, a transient hypoxia stimulus was applied to induce a bolus of deoxyhemoglobins as an endogenous paramagnetic contrast in blood oxygenation level-dependent (BOLD) MRI. Based on stimulus-duration-dependent studies, 5 s anoxic stimulus was chosen, which induced a decrease in arterial oxygenation to 59%. Dynamic susceptibility changes were acquired with whole-brain BOLD MRI using both all-vessel-sensitive gradient-echo and microvascular-sensitive spin-echo readouts. Cerebral blood flow (CBF) and cerebral blood volume (CBV) were quantified by modeling BOLD dynamics using a partial-volume-corrected arterial input function. In the mouse under ketamine/xylazine anesthesia, total CBF and CBV were 112.0 ± 15.0 ml/100 g/min and 3.39 ± 0.59 ml/100 g (n = 15 mice), respectively, whereas microvascular CBF and CBV were 85.8 ± 6.9 ml/100 g/min and 2.23 ± 0.27 ml/100 g (n = 7 mice), respectively. Regional total vs. microvascular perfusion metrics were highly correlated but a slight mismatch was observed in the large-vessel areas and cortical depth profiles. Overall, this non-invasive, repeatable, simple hypoxia BOLD-MRI approach is viable for perfusion mapping of rodents.

Theory of chemical exchange saturation transfer MRI in the context of different magnetic fields.

Chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) is a versatile MRI method that provides contrast based on the level of molecular and metabolic activity. This contrast arises from indirect measurement of protons in low concentration molecules that are exchanging with the abundant water proton pool. The indirect measurement is based on magnetization transfer of radio frequency (rf)-prepared magnetization from the small pool to the water pool. The signal can be modeled by the Bloch-McConnell equations combining standard magnetization dynamics and chemical exchange processes. In this article, we review analytical solutions of the Bloch-McConnell equations and especially the derived CEST signal equations and their implications. The analytical solutions give direct insight into the dependency of measurable CEST effects on underlying parameters such as the exchange rate and concentration of the solute pools, but also on the system parameters such as the rf irradiation field B1 , as well as the static magnetic field B0 . These theoretical field-strength dependencies and their influence on sequence design are highlighted herein. In vivo results of different groups making use of these field-strength benefits/dependencies are reviewed and discussed.