Combining CRISPR-Cas9 and brain imaging to study the link from genes to molecules to networks.

Receptors, transporters, and ion channels are important targets for therapy development in neurological diseases, but their mechanistic role in pathogenesis is often poorly understood. Gene editing and in vivo imaging approaches will help to identify the molecular and functional role of these targets and the consequence of their regional dysfunction on the whole-brain level. We combine CRISPR-Cas9 gene editing with in vivo positron emission tomography (PET) and functional MRI (fMRI) to investigate the direct link between genes, molecules, and the brain connectome. The extensive knowledge of the Slc18a2 gene encoding the vesicular monoamine transporter (VMAT2), involved in the storage and release of dopamine, makes it an excellent target for studying the gene network relationships while structurally preserving neuronal integrity and function. We edited the Slc18a2 in the substantia nigra pars compacta of adult rats and used in vivo molecular imaging besides behavioral, histological, and biochemical assessments to characterize the CRISPR-Cas9-mediated VMAT2 knockdown. Simultaneous PET/fMRI was performed to investigate molecular and functional brain alterations. We found that stage-specific adaptations of brain functional connectivity follow the selective impairment of presynaptic dopamine storage and release. Our study reveals that recruiting different brain networks is an early response to the dopaminergic dysfunction preceding neuronal cell loss. Our combinatorial approach is a tool to investigate the impact of specific genes on brain molecular and functional dynamics, which will help to develop tailored therapies for normalizing brain function.

Western diet increases brain metabolism and adaptive immune responses in a mouse model of amyloidosis.

Diet-induced increase in body weight is a growing health concern worldwide. Often accompanied by a low-grade metabolic inflammation that changes systemic functions, diet-induced alterations may contribute to neurodegenerative disorder progression as well. This study aims to non-invasively investigate diet-induced metabolic and inflammatory effects in the brain of an APPPS1 mouse model of Alzheimer's disease. [18F]FDG, [18F]FTHA, and [18F]GE-180 were used for in vivo PET imaging in wild-type and APPPS1 mice. Ex vivo flow cytometry and histology in brains complemented the in vivo findings. 1H- magnetic resonance spectroscopy in the liver, plasma metabolomics and flow cytometry of the white adipose tissue were used to confirm metaflammatory condition in the periphery. We found disrupted glucose and fatty acid metabolism after Western diet consumption, with only small regional changes in glial-dependent neuroinflammation in the brains of APPPS1 mice. Further ex vivo investigations revealed cytotoxic T cell involvement in the brains of Western diet-fed mice and a disrupted plasma metabolome. 1H-magentic resonance spectroscopy and immunological results revealed diet-dependent inflammatory-like misbalance in livers and fatty tissue. Our multimodal imaging study highlights the role of the brain-liver-fat axis and the adaptive immune system in the disruption of brain homeostasis in amyloid models of Alzheimer's disease.

Elucidating the complementarity of resting-state networks derived from dynamic [18F]FDG and hemodynamic fluctuations using simultaneous small-animal PET/MRI.

Functional connectivity (FC) and resting-state network (RSN) analyses using functional magnetic resonance imaging (fMRI) have evolved into a growing field of research and have provided useful biomarkers for the assessment of brain function in neurological disorders. However, the underlying mechanisms of the blood oxygen level-dependant (BOLD) signal are not fully resolved due to its inherent complexity. In contrast, [18F]fluorodeoxyglucose positron emission tomography ([18F]FDG-PET) has been shown to provide a more direct measure of local synaptic activity and may have additional value for the readout and interpretation of brain connectivity. We performed an RSN analysis from simultaneously acquired PET/fMRI data on a single-subject level to directly compare fMRI and [18F]FDG-PET-derived networks during the resting state. Simultaneous [18F]FDG-PET/fMRI scans were performed in 30 rats. Pairwise correlation analysis, as well as independent component analysis (ICA), were used to compare the readouts of both methods. We identified three RSNs with a high degree of similarity between PET and fMRI-derived readouts: the default-mode-like network (DMN), the basal ganglia network and the cerebellar-midbrain network. Overall, [18F]FDG connectivity indicated increased integration between different, often distant, brain areas compared to the results indicated by the more segregated fMRI-derived FC. Additionally, several networks exclusive to either modality were observed using ICA. These networks included mainly bilateral cortical networks of a limited spatial extent for fMRI and more spatially widespread networks for [18F]FDG-PET, often involving several subcortical areas. This is the first study using simultaneous PET/fMRI to report RSNs subject-wise from dynamic [18F]FDG tracer delivery and BOLD fluctuations with both independent component analysis (ICA) and pairwise correlation analysis in small animals. Our findings support previous studies, which show a close link between local synaptic glucose consumption and BOLD-fMRI-derived FC. However, several brain regions were exclusively attributed to either [18F]FDG or BOLD-derived networks underlining the complementarity of this hybrid imaging approach, which may contribute to the understanding of brain functional organization and could be of interest for future clinical applications.

Striatal and prefrontal D2R and SERT distributions contrastingly correlate with default-mode connectivity.

Although brain research has taken important strides in recent decades, the interaction and coupling of its different physiological levels is still not elucidated. Specifically, the molecular substrates of resting-state functional connectivity (rs-FC) remain poorly understood. The aim of this study was elucidating interactions between dopamine D2 receptors (D2R) and serotonin transporter (SERT) availabilities in the striatum (CPu) and medial prefrontal cortex (mPFC), two of the main dopaminergic and serotonergic projection areas, and the default-mode network. Additionally, we delineated its interaction with two other prominent resting-state networks (RSNs), the salience network (SN) and the sensorimotor network (SMN). To this extent, we performed simultaneous PET/fMRI scans in a total of 59 healthy rats using [11C]raclopride and [11C]DASB, two tracers used to image quantify D2R and SERT respectively. Edge, node and network-level rs-FC metrics were calculated for each subject and potential correlations with binding potentials (BPND) in the CPu and mPFC were evaluated. We found widespread negative associations between CPu D2R availability and all the RSNs investigated, consistent with the postulated role of the indirect basal ganglia pathway. Correlations between D2Rs in the mPFC were weaker and largely restricted to DMN connectivity. Strikingly, medial prefrontal SERT correlated both positively with anterior DMN rs-FC and negatively with rs-FC between and within the SN, SMN and the posterior DMN, underlining the complex role of serotonergic neurotransmission in this region. Here we show direct relationships between rs-FC and molecular properties of the brain as assessed by simultaneous PET/fMRI in healthy rodents. The findings in the present study contribute to the basic understanding of rs-FC by revealing associations between inter-subject variances of rs-FC and receptor and transporter availabilities. Additionally, since current therapeutic strategies typically target neurotransmitter systems with the aim of normalizing brain function, delineating associations between molecular and network-level brain properties is essential and may enhance the understanding of neuropathologies and support future drug development.

Functional resting-state brain connectivity is accompanied by dynamic correlations of application-dependent [18F]FDG PET-tracer fluctuations.

Brain function is characterized by a convolution of various biochemical and physiological processes, raising the interest whether resting-state functional connectivity derived from hemodynamic scales shows underlying metabolic synchronies. Increasing evidence suggests that metabolic connectivity based on glucose consumption associated PET recordings may serve as a marker of cognitive functions and neuropathologies. However, to what extent fMRI-derived resting-state brain connectivity can also be characterized based on dynamic fluctuations of glucose metabolism and how metabolic connectivity is influenced by [18F]FDG pharmacokinetics remains unsolved. Simultaneous PET/MRI measurements were performed in a total of 26 healthy male Lewis rats. Simultaneously to resting-state fMRI scans, one cohort (n = 15) received classical bolus [18F]FDG injections and dynamic PET images were recorded. In a second cohort (n = 11) [18F]FDG was constantly infused over the entire functional PET/MRI scans. Resting-state fMRI and [18F]FDG-PET connectivity was evaluated using a graph-theory based correlation approach and compared on whole-brain level and for a default-mode network-like structure. Further, pharmacokinetic and tracer uptake influences on [18F]FDG-PET connectivity results were investigated based on the different PET protocols. By integrating simultaneous resting-state fMRI and dynamic [18F]FDG-PET measurements in the rat brain, we identified homotopic correlations between both modalities, suggesting an underlying synchrony between hemodynamic processes and glucose consumption. Furthermore, the presence of the prominent resting-state default-mode network-like structure was not only depicted on a functional scale but also from dynamic fluctuations of [18F]FDG. In addition, the present findings demonstrated strong pharmacokinetic and tracer uptake dependencies of [18F]FDG-PET connectivity outcomes. This study highlights the application of dynamic [18F]FDG-PET to study cognitive brain functions and to decode underlying brain networks in the resting-state. Thereby, PET-derived connectivity outcomes indicated strong dependencies on tracer application regimens and subsequent time-varying tracer pharmacokinetics.

Interregional causal influences of brain metabolic activity reveal the spread of aging effects during normal aging.

During healthy brain aging, different brain regions show anatomical or functional declines at different rates, and some regions may show compensatory increases in functional activity. However, few studies have explored interregional influences of brain activity during the aging process. We proposed a causality analysis framework combining high dimensionality independent component analysis (ICA), Granger causality, and least absolute shrinkage and selection operator regression on longitudinal brain metabolic activity data measured by Fludeoxyglucose positron emission tomography (FDG-PET). We analyzed FDG-PET images from healthy old subjects, who were scanned for at least five sessions with an averaged intersession interval of 1 year. The longitudinal data were concatenated across subjects to form a time series, and the first-order autoregressive model was used to measure interregional causality among the independent sources of metabolic activity identified using ICA. Several independent sources with reduced metabolic activity in aging, including the anterior temporal lobe and orbital frontal cortex, demonstrated causal influences over many widespread brain regions. On the other hand, the influenced regions were more distributed, and had smaller age-related declines or even relatively increased metabolic activity. The current data demonstrated interregional spreads of aging on metabolic activity at the scale of a year, and have identified key brain regions in the aging process that have strong influences over other regions.

Functional PET/MRI reveals active inhibition of neuronal activity during optogenetic activation of the nigrostriatal pathway.

The dopaminergic system is a central component of the brain's neurobiological framework, governing motor control and reward responses and playing an essential role in various brain disorders. Within this complex network, the nigrostriatal pathway represents a critical circuit for dopamine neurotransmission from the substantia nigra to the striatum. However, stand-alone functional magnetic resonance imaging is unable to study the intricate interplay between brain activation and its molecular underpinnings. In our study, the use of a functional [fluorine-18]2-fluor-2-deoxy-d-glucose positron emission tomography approach, simultaneously with blood oxygen level-dependent functional magnetic resonance imaging, provided an important insight that demonstrates an active suppression of the nigrostriatal activity during optogenetic stimulation. This result increases our understanding of the molecular mechanisms of brain function and provides an important perspective on how dopamine influences hemodynamic responses in the brain.

Long-term adaptation of prefrontal circuits in a mouse model of NMDAR hypofunction.

Pharmacological approaches to induce N-methyl-d-aspartate receptor (NMDAR) hypofunction have been intensively used to understand the aetiology and pathophysiology of schizophrenia. Yet, the precise cellular and molecular mechanisms that relate to brain network dysfunction remain largely unknown. Here, we used a set of complementary approaches to assess the functional network abnormalities present in male mice that underwent a 7-day subchronic phencyclidine (PCP 10 mg/kg, subcutaneously, once daily) treatment. Our data revealed that pharmacological intervention with PCP affected cognitive performance and auditory evoked gamma oscillations in the prefrontal cortex (PFC) mimicking endophenotypes of some schizophrenia patients. We further assessed PFC cellular function and identified altered neuronal intrinsic membrane properties, reduced parvalbumin (PV) immunostaining and diminished inhibition onto L5 PFC pyramidal cells. A decrease in the strength of optogenetically-evoked glutamatergic current at the ventral hippocampus to PFC synapse was also demonstrated, along with a weaker shunt of excitatory transmission by local PFC interneurons. On a macrocircuit level, functional ultrasound measurements indicated compromised functional connectivity within several brain regions particularly involving PFC and frontostriatal circuits. Herein, we reproduced a panel of schizophrenia endophenotypes induced by subchronic PCP application in mice. We further recapitulated electrophysiological signatures associated with schizophrenia and provided an anatomical reference to critical elements in the brain circuitry. Together, our findings contribute to a better understanding of the physiological underpinnings of deficits induced by subchronic NMDAR antagonist regimes and provide a test system for characterization of pharmacological compounds.

Functional ultrasound detects frequency-specific acute and delayed S-ketamine effects in the healthy mouse brain.

Introduction: S-ketamine has received great interest due to both its antidepressant effects and its potential to induce psychosis when administered subchronically. However, no studies have investigated both its acute and delayed effects using in vivo small-animal imaging. Recently, functional ultrasound (fUS) has emerged as a powerful alternative to functional magnetic resonance imaging (fMRI), outperforming it in sensitivity and in spatiotemporal resolution. In this study, we employed fUS to thoroughly characterize acute and delayed S-ketamine effects on functional connectivity (FC) within the same cohort at slow frequency bands ranging from 0.01 to 1.25 Hz, previously reported to exhibit FC. Methods: We acquired fUS in a total of 16 healthy C57/Bl6 mice split in two cohorts (n = 8 received saline, n = 8 S-ketamine). One day after the first scans, performed at rest, the mice received the first dose of S-ketamine during the second measurement, followed by four further doses administered every 2 days. First, we assessed FC reproducibility and reliability at baseline in six frequency bands. Then, we investigated the acute and delayed effects at day 1 after the first dose and at day 9, 1 day after the last dose, for all bands, resulting in a total of four fUS measurements for every mouse. Results: We found reproducible (r > 0.9) and reliable (r > 0.9) group-average readouts in all frequency bands, only the 0.01-0.27 Hz band performing slightly worse. Acutely, S-ketamine induced strong FC increases in five of the six bands, peaking in the 0.073-0.2 Hz band. These increases comprised both cortical and subcortical brain areas, yet were of a transient nature, FC almost returning to baseline levels towards the end of the scan. Intriguingly, we observed robust corticostriatal FC decreases in the fastest band acquired (0.75 Hz-1.25 Hz). These changes persisted to a weaker extent after 1 day and at this timepoint they were accompanied by decreases in the other five bands as well. After 9 days, the decreases in the 0.75-1.25 Hz band were maintained, however no changes between cohorts could be detected in any other bands. Discussion: In summary, the study reports that acute and delayed ketamine effects in mice are not only dissimilar but have different directionalities in most frequency bands. The complementary readouts of the employed frequency bands recommend the use of fUS for frequency-specific investigation of pharmacological effects on FC.

Neurovascular Uncoupling: Multimodal Imaging Delineates the Acute Effects of 3,4-Methylenedioxymethamphetamine.

Psychedelic compounds such as 3,4-methylenedioxymethamphetamine (MDMA) have attracted increasing interest in recent years because of their therapeutic potential in psychiatric disorders. To understand the acute effects of psychedelic drugs in vivo, blood-oxygenation-level-dependent (BOLD) functional MRI (fMRI) has been widely used. In particular, fMRI studies have suggested that MDMA leads to inhibition of brain activity, challenging previous hypotheses indicating mainly excitatory effects based, among others, on increased metabolism shown by 18F-FDG functional PET (fPET). However, interpretation of hemodynamic changes induced by psychedelics is difficult because of their potent vascular effects. Methods: We aimed to delineate the acute effects of MDMA using simultaneous PET/fMRI in rats. For this purpose, hemodynamic changes measured by BOLD fMRI were related to alterations in glucose utilization and serotonin transporter (SERT) occupancy using 18F-FDG fPET/fMRI and 11C-DASB PET/fMRI. Results: We show that MDMA induces localized increases in glucose metabolism in limbic projection areas involved in emotional processing. The increased glucose metabolism was accompanied by global cerebral and extracerebral hemodynamic decreases. We further demonstrated a strong correlation between SERT occupancies and regional BOLD reductions after acute MDMA administration. Conclusion: Our data indicate that hemodynamic decreases after acute MDMA administration are of a nonneuronal nature and initiate peripherally. Within the brain, MDMA triggers neuronal activation in limbic projection areas, whereas increased serotonin levels induced by SERT blockage cause neurovascular uncoupling through direct vascular effects. Correct understanding of the in vivo mechanism of MDMA not only supports ongoing research but also warrants a reassessment of previous studies on neuronal effects of psychedelics relying on neurovascular coupling and recommends 18F-FDG fPET as a potentially more robust measure for pharmacologic research.

A consensus protocol for functional connectivity analysis in the rat brain.

Task-free functional connectivity in animal models provides an experimental framework to examine connectivity phenomena under controlled conditions and allows for comparisons with data modalities collected under invasive or terminal procedures. Currently, animal acquisitions are performed with varying protocols and analyses that hamper result comparison and integration. Here we introduce StandardRat, a consensus rat functional magnetic resonance imaging acquisition protocol tested across 20 centers. To develop this protocol with optimized acquisition and processing parameters, we initially aggregated 65 functional imaging datasets acquired from rats across 46 centers. We developed a reproducible pipeline for analyzing rat data acquired with diverse protocols and determined experimental and processing parameters associated with the robust detection of functional connectivity across centers. We show that the standardized protocol enhances biologically plausible functional connectivity patterns relative to previous acquisitions. The protocol and processing pipeline described here is openly shared with the neuroimaging community to promote interoperability and cooperation toward tackling the most important challenges in neuroscience.