Impaired consciousness in temporal lobe seizures has a major negative impact on quality of life. The prevailing view holds that this disorder impairs consciousness by seizure spread to the bilateral temporal lobes. We propose instead that seizures invade subcortical regions and depress arousal, causing impairment through decreases rather than through increases in activity. Using functional magnetic resonance imaging in a rodent model, we found increased activity in regions known to depress cortical function, including lateral septum and anterior hypothalamus. Importantly, we found suppression of intralaminar thalamic and brainstem arousal systems and suppression of the cortex. At a cellular level, we found reduced firing of identified cholinergic neurons in the brainstem pedunculopontine tegmental nucleus and basal forebrain. Finally, we used enzyme-based amperometry to demonstrate reduced cholinergic neurotransmission in both cortex and thalamus. Decreased subcortical arousal is a critical mechanism for loss of consciousness in focal temporal lobe seizures.

Functional magnetic resonance imaging (fMRI) techniques using the blood-oxygen level-dependent (BOLD) signal have shown great potential as clinical biomarkers of disease. Thus, using these techniques in preclinical rodent models is an urgent need. Calibrated fMRI is a promising technique that can provide high-resolution mapping of cerebral oxygen metabolism (CMRO2). However, calibrated fMRI is difficult to use in rodent models for several reasons: rodents are anesthetized, stimulation-induced changes are small, and gas challenges induce noisy CMRO2 predictions. We used, in mice, a relaxometry-based calibrated fMRI method which uses cerebral blood flow (CBF) and the BOLD-sensitive magnetic relaxation component, R2', the same parameter derived in the deoxyhemoglobin-dilution model of calibrated fMRI. This method does not use any gas challenges, which we tested on mice in both awake and anesthetized states. As anesthesia induces a whole-brain change, our protocol allowed us to overcome the former limitations of rodent studies using calibrated fMRI. We revealed 1.5-2 times higher CMRO2, dependent upon brain region, in the awake state versus the anesthetized state. Our results agree with alternative measurements of whole-brain CMRO2 in the same mice and previous human anesthesia studies. The use of calibrated fMRI in rodents has much potential for preclinical fMRI.

Neural tube defects are a common congenital anomaly involving incomplete closure of the spinal cord. Myelomeningocele (MMC) is a severe form in which there is complete exposure of neural tissue with a lack of skin, soft tissue, or bony covering to protect the spinal cord. The all-trans retinoic acid (ATRA) induced rat model of (MMC) is a reproducible, cost-effective means of studying this disease; however, there are limited modalities to objectively quantify disease severity, or potential benefits from experimental therapies. We sought to determine the feasibility of detecting differences between MMC and wild type (WT) rat fetuses using diffusion magnetic resonance imaging techniques (MRI). Rat dams were gavage-fed ATRA to produce MMC defects in fetuses, which were surgically delivered prior to term. Average diffusion coefficient (ADC) and fractional anisotropy (FA) maps were obtained for each fetus. Brain volumes and two anatomically defined brain length measurements (D1 and D2) were significantly decreased in MMC compared to WT. Mean ADC signal was significantly increased in MMC compared to WT, but no difference was found for FA signal. In summary, ADC and brain measurements were significantly different between WT and MMC rat fetuses. ADC could be a useful complementary imaging biomarker to current histopathologic analysis of MMC models, and potentially expedite therapeutic research for this disease.

The investigation of neural circuits is important for interpreting both healthy brain function and psychiatric disorders. Currently, the architecture of neural circuits is always investigated with fluorescent protein encoding neurotropic virus and ex vivo fluorescent imaging technology. However, it is difficult to obtain a whole-brain neural circuit connection in living animals, due to the limited fluorescent imaging depth. Herein, the noninvasive, whole-brain imaging technique of MRI and the hypotoxicity virus vector AAV (adeno-associated virus) were combined to investigate the whole-brain neural circuits in vivo. AAV2-retro are an artificially-evolved virus vector that permits access to the terminal of neurons and retrograde transport to their cell bodies. By expressing the ferritin protein which could accumulate iron ions and influence the MRI contrast, the neurotropic virus can cause MRI signal changes in the infected regions. For mice injected with the ferritin-encoding virus vector (rAAV2-retro-CAG-Ferritin) in the caudate putamen (CPu), several regions showed significant changes in MRI contrasts, such as PFC (prefrontal cortex), HIP (hippocampus), Ins (insular cortex) and BLA (basolateral amygdala). The expression of ferritin in those regions was also verified with ex vivo fluorescence imaging. In addition, we demonstrated that changes in T2 relaxation time could be used to identify the spread area of the virus in the brain over time. Thus, the neural connections could be longitudinally detected with the in vivo MRI method. This novel technique could be utilized to observe the viral infection process and detect the neural circuits in a living animal.

Under normal conditions, high sodium (Na+) in extracellular (Na+e) and blood (Na+b) compartments and low Na+ in intracellular milieu (Na+i) produce strong transmembrane (ΔNa+mem) and weak transendothelial (ΔNa+end) gradients respectively, and these manifest the cell membrane potential (Vm) as well as blood-brain barrier (BBB) integrity. We developed a sodium (23Na) magnetic resonance spectroscopic imaging (MRSI) method using an intravenously-administered paramagnetic polyanionic agent to measure ΔNa+mem and ΔNa+end. In vitro 23Na-MRSI established that the 23Na signal is intensely shifted by the agent compared to other biological factors (e.g., pH and temperature). In vivo 23Na-MRSI showed Na+i remained unshifted and Na+b was more shifted than Na+e, and these together revealed weakened ΔNa+mem and enhanced ΔNa+end in rat gliomas (vs. normal tissue). Compared to normal tissue, RG2 and U87 tumors maintained weakened ΔNa+mem (i.e., depolarized Vm) implying an aggressive state for proliferation, whereas RG2 tumors displayed elevated ∆Na+end suggesting altered BBB integrity. We anticipate that 23Na-MRSI will allow biomedical explorations of perturbed Na+ homeostasis in vivo.

The thalamus is a crucial subcortical hub that impacts cortical activity. Tracing experiments in animals and post-mortem humans suggest rich morphological specificity of the thalamus. Very few studies reported rodent thalamic activations by functional MRI (fMRI) as compared to cortical activations for different sensory stimuli. Here, we show different portions of the rat thalamus in response to tactile (forepaw, whisker) and non-tactile (visual, olfactory) sensory stimuli with high field fMRI (11.7T) using a custom-build quadrature surface coil to capture high sensitivity signals from superficial and deep brain regions simultaneously. Results demonstrate reproducible thalamic activations during both tactile and non-tactile stimuli. Forepaw and whisker stimuli activated broader regions within the thalamus: ventral posterior lateral (VPL), ventral posterior medial (VPM), lateral posterior mediorostral (LPMR) and posterior medial (POm) thalamic nuclei. Visual stimuli activated dorsal lateral geniculate nucleus (DLG) of the thalamus but also parts of the superior/inferior colliculus, whereas olfactory stimuli activated specifically the mediodorsal nucleus of the thalamus (MDT). BOLD activations in LGN and MDT were much stronger than in VPL, VPM, LPMR and POm. These fMRI-based thalamic activations suggest that forepaw and whisker (i.e., tactile) stimuli engage VPL, VPM, LPMR and POm whereas visual and olfactory (i.e., non-tactile) stimuli, respectively, recruit DLG and MDT exclusively.

Human cortical organoids (hCOs), derived from human embryonic stem cells (hESCs), provide a platform to study human brain development and diseases in complex three-dimensional tissue. However, current hCOs lack microvasculature, resulting in limited oxygen and nutrient delivery to the inner-most parts of hCOs. We engineered hESCs to ectopically express human ETS variant 2 (ETV2). ETV2-expressing cells in hCOs contributed to forming a complex vascular-like network in hCOs. Importantly, the presence of vasculature-like structures resulted in enhanced functional maturation of organoids. We found that vascularized hCOs (vhCOs) acquired several blood-brain barrier characteristics, including an increase in the expression of tight junctions, nutrient transporters and trans-endothelial electrical resistance. Finally, ETV2-induced endothelium supported the formation of perfused blood vessels in vivo. These vhCOs form vasculature-like structures that resemble the vasculature in early prenatal brain, and they present a robust model to study brain disease in vitro.

To demonstrate feasibility of developing a noninvasive extracellular pH (pHe ) mapping method on a clinical MRI scanner for molecular imaging of liver cancer.

Based on the premise that physical activity/exercise impacts hippocampal structure and function, we investigated if hippocampal metabolites for neuronal viability and cell membrane density (i.e., N-acetyl aspartate (NAA), choline (Cho), creatine (Cr)) were higher in older adults performing supervised exercise compared to following national physical activity guidelines. Sixty-three participants (75.3 ± 1.9 years after 3 years of intervention) recruited from the Generation 100 study (NCT01666340_date:08.16.2012) were randomized into a supervised exercise group (SEG) performing twice weekly moderate- to high-intensity training, and a control group (CG) following national physical activity guidelines of ≥ 30-min moderate physical activity ≥ 5 days/week. Hippocampal body and head volumes and NAA, Cho, and Cr levels were acquired at 3T with magnetic resonance imaging and spectroscopic imaging. Sociodemographic data, peak oxygen uptake (VO2peak), exercise characteristics, psychological health, and cognition were recorded. General linear models were used to assess group differences and associations corrected for age, sex, education, and hippocampal volume. Both groups adhered to their training, where SEG trained at higher intensity. SEG had significantly lower NAA/Cr in hippocampal body than CG (p = 0.04). Across participants, higher training intensity was associated with lower Cho/Cr in hippocampal body (p < 0.001). Change in VO2peak, increasing VO2peak from baseline to 3 years, or VO2peak at 3 years were not associated with hippocampal neurochemicals. Lower NAA/Cr in hippocampal body was associated with poorer psychological health and slightly higher cognitive scores. Thus, following the national physical activity guidelines and not training at the highest intensity level were associated with the best neurochemical profile in the hippocampus at 3 years.

Absence seizures are brief episodes of impaired consciousness, behavioral arrest, and unresponsiveness, with yet-unknown neuronal mechanisms. Here we report that an awake female rat model recapitulates the behavioral, electroencephalographic, and cortical functional magnetic resonance imaging characteristics of human absence seizures. Neuronally, seizures feature overall decreased but rhythmic firing of neurons in cortex and thalamus. Individual cortical and thalamic neurons express one of four distinct patterns of seizure-associated activity, one of which causes a transient initial peak in overall firing at seizure onset, and another which drives sustained decreases in overall firing. 40-60 s before seizure onset there begins a decline in low frequency electroencephalographic activity, neuronal firing, and behavior, but an increase in higher frequency electroencephalography and rhythmicity of neuronal firing. Our findings demonstrate that prolonged brain state changes precede consciousness-impairing seizures, and that during seizures distinct functional groups of cortical and thalamic neurons produce an overall transient firing increase followed by a sustained firing decrease, and increased rhythmicity.

RAC1P29S is the third most prevalent hotspot mutation in sun-exposed melanoma. RAC1 alterations in cancer are correlated with poor prognosis, resistance to standard chemotherapy, and insensitivity to targeted inhibitors. Although RAC1P29S mutations in melanoma and RAC1 alterations in several other cancers are increasingly evident, the RAC1-driven biological mechanisms contributing to tumorigenesis remain unclear. Lack of rigorous signaling analysis has prevented identification of alternative therapeutic targets for RAC1P29S-harboring melanomas. To investigate the RAC1P29S-driven effect on downstream molecular signaling pathways, we generated an inducible RAC1P29S expression melanocytic cell line and performed RNA-sequencing (RNA-seq) coupled with multiplexed kinase inhibitor beads and mass spectrometry (MIBs/MS) to establish enriched pathways from the genomic to proteomic level. Our proteogenomic analysis identified CDK9 as a potential new and specific target in RAC1P29S-mutant melanoma cells. In vitro, CDK9 inhibition impeded the proliferation of in RAC1P29S-mutant melanoma cells and increased surface expression of PD-L1 and MHC Class I proteins. In vivo, combining CDK9 inhibition with anti-PD-1 immune checkpoint blockade significantly inhibited tumor growth only in melanomas that expressed the RAC1P29S mutation. Collectively, these results establish CDK9 as a novel target in RAC1-driven melanoma that can further sensitize the tumor to anti-PD-1 immunotherapy.