pNet: A toolbox for personalized functional networks modeling.

Personalized functional networks (FNs) derived from functional magnetic resonance imaging (fMRI) data are useful for characterizing individual variations in the brain functional topography associated with the brain development, aging, and disorders. To facilitate applications of the personalized FNs with enhanced reliability and reproducibility, we develop an open-source toolbox that is user-friendly, extendable, and includes rigorous quality control (QC), featuring multiple user interfaces (graphics, command line, and a step-by-step guideline) and job-scheduling for high performance computing (HPC) clusters. Particularly, the toolbox, named personalized functional network modeling (pNet), takes fMRI inputs in either volumetric or surface type, ensuring compatibility with multiple fMRI data formats, and computes personalized FNs using two distinct modeling methods: one method optimizes the functional coherence of FNs, while the other enhances their independence. Additionally, the toolbox provides HTML-based reports for QC and visualization of personalized FNs. The toolbox is developed in both MATLAB and Python platforms with a modular design to facilitate extension and modification by users familiar with either programming language. We have evaluated the toolbox on two fMRI datasets and demonstrated its effectiveness and user-friendliness with interactive and scripting examples. pNet is publicly available at https://github.com/MLDataAnalytics/pNet.

Suppressing Anterior Cingulate Cortex Modulates Default Mode Network and Behavior in Awake Rats.

The default mode network (DMN) is a principal brain network in the mammalian brain. Although the DMN in humans has been extensively studied with respect to network structure, function, and clinical implications, our knowledge of DMN in animals remains limited. In particular, the functional role of DMN nodes, and how DMN organization relates to DMN-relevant behavior are still elusive. Here we investigated the causal relationship of inactivating a pivotal node of DMN (i.e., dorsal anterior cingulate cortex [dACC]) on DMN function, network organization, and behavior by combining chemogenetics, resting-state functional magnetic resonance imaging (rsfMRI) and behavioral tests in awake rodents. We found that suppressing dACC activity profoundly changed the activity and connectivity of DMN, and these changes were associated with altered DMN-related behavior in animals. The chemo-rsfMRI-behavior approach opens an avenue to mechanistically dissecting the relationships between a specific node, brain network function, and behavior. Our data suggest that, like in humans, DMN in rodents is a functional network with coordinated activity that mediates behavior.

miR-16 exhibits protective function in LPS-treated cardiomyocytes by targeting DOCK2 to repress cell apoptosis and exert anti-inflammatory effect.

This study aims to investigate the effects of microRNA (miR)-16/dedicator of cytokinesis 2 (DOCK2) on myocarditis. The differences in the expression of genes in acute myocarditis were filtered out across Gene Expression Omnibus (GEO) database. Myocarditis cell model was established by lipopolysaccharide (LPS) stimulation in cardiomyocytes. The association between miR-16 and DOCK2 was predicted by bioinformatics software and confirmed by dual-luciferase assay. Polymerase chain reaction and western blot analysis were employed to assess the expression levels of miR-16 and DOCK2 under different conditions. Cells viability, apoptosis, and inflammatory reaction were evaluated by Cell Counting Kit-8, flow cytometry, and enzyme-linked immunosorbent assays. miR-16, as an upstream regulator of DOCK2, exhibited lower expression in LPS-induced myocarditis model. More importantly, we revealed that a marked augmentation of miR-16 promoted the growth of LPS-stimulated cardiomyocytes, and attenuated cell apoptosis and inflammatory response. However, an increasing expression of DOCK2 inhibited the remission of LPS-induced myocardial injury caused by miR-16 mimic. Herein, our results highlighted that upregulation of miR-16 resulted in the protective effects on LPS-induced myocardial injury by reducing DOCK2 expression, affording a pair of novel target molecules for ameliorating the symptoms of myocarditis.

Global brain signal in awake rats.

Although often used as a nuisance in resting-state functional magnetic resonance imaging (rsfMRI), the global brain signal in humans and anesthetized animals has important neural basis. However, our knowledge of the global signal in awake rodents is sparse. To bridge this gap, we systematically analyzed rsfMRI data acquired with a conventional single-echo (SE) echo planar imaging (EPI) sequence in awake rats. The spatial pattern of rsfMRI frames during peaks of the global signal exhibited prominent co-activations in the thalamo-cortical and hippocampo-cortical networks, as well as in the basal forebrain, hinting that these neural networks might contribute to the global brain signal in awake rodents. To validate this concept, we acquired rsfMRI data using a multi-echo (ME) EPI sequence and removed non-neural components in the rsfMRI signal. Consistent co-activation patterns were obtained in extensively de-noised ME-rsfMRI data, corroborating the finding from SE-rsfMRI data. Furthermore, during rsfMRI experiments, we simultaneously recorded neural spiking activities in the hippocampus using GCaMP-based fiber photometry. The hippocampal calcium activity exhibited significant correspondence with the global rsfMRI signal. These data collectively suggest that the global rsfMRI signal contains significant neural components that involve coordinated activities in the thalamo-cortical and hippocampo-cortical networks. These results provide important insight into the neural substrate of the global brain signal in awake rodents.

Individual variability in behavior and functional networks predicts vulnerability using an animal model of PTSD.

Only a minority of individuals experiencing trauma subsequently develop post-traumatic stress disorder (PTSD). However, whether differences in vulnerability to PTSD result from a predisposition or trauma exposure remains unclear. A major challenge in differentiating these possibilities is that clinical studies focus on individuals already exposed to trauma without pre-trauma conditions. Here, using the predator scent model of PTSD in rats and a longitudinal design, we measure pre-trauma brain-wide neural circuit functional connectivity, behavioral and corticosterone responses to trauma exposure, and post-trauma anxiety. Freezing during predator scent exposure correlates with functional connectivity in a set of neural circuits, indicating pre-existing circuit function can predispose animals to differential fearful responses to threats. Counterintuitively, rats with lower freezing show more avoidance of the predator scent, a prolonged corticosterone response, and higher anxiety long after exposure. This study provides a framework of pre-existing circuit function that determines threat responses, which might directly relate to PTSD-like behaviors.

Development of brain-wide connectivity architecture in awake rats.

Childhood and adolescence are both critical developmental periods, evidenced by complex neurophysiological changes the brain undergoes and high occurrence rates of neuropsychiatric disorders during these periods. Despite substantial progress in elucidating the developmental trajectories of individual neural circuits, our knowledge of developmental changes of whole-brain connectivity architecture in animals is sparse. To fill this gap, here we longitudinally acquired rsfMRI data in awake rats during five developmental stages from juvenile to adulthood. We found that the maturation timelines of brain circuits were heterogeneous and system specific. Functional connectivity (FC) tended to decrease in subcortical circuits, but increase in cortical circuits during development. In addition, the developing brain exhibited hemispheric functional specialization, evidenced by reduced inter-hemispheric FC between homotopic regions, and lower similarity of region-to-region FC patterns between the two hemispheres. Finally, we showed that whole-brain network development was characterized by reduced clustering (i.e. local communication) but increased integration (distant communication). Taken together, the present study has systematically characterized the development of brain-wide connectivity architecture from juvenile to adulthood in awake rats. It also serves as a critical reference point for understanding circuit- and network-level changes in animal models of brain development-related disorders. Furthermore, FC data during brain development in awake rodents contain high translational value and can shed light onto comparative neuroanatomy.

Simultaneous GCaMP6-based fiber photometry and fMRI in rats.

BACKGROUND: Understanding the relationship between neural and vascular signals is essential for interpretation of functional MRI (fMRI) results with respect to underlying neuronal activity. Simultaneously measuring neural activity using electrophysiology with fMRI has been highly valuable in elucidating the neural basis of the blood oxygenation-level dependent (BOLD) signal. However, this approach is also technically challenging due to the electromagnetic interference that is observed in electrophysiological recordings during MRI scanning. NEW METHOD: Recording optical correlates of neural activity, such as calcium signals, avoids this issue, and has opened a new avenue to simultaneously acquire neural and BOLD signals. RESULTS: The present study is the first to demonstrate the feasibility of simultaneously and repeatedly acquiring calcium and BOLD signals in animals using a genetically encoded calcium indicator, GCaMP6. This approach was validated with a visual stimulation experiment, during which robust increases of both calcium and BOLD signals in the superior colliculus were observed. In addition, repeated measurement in the same animal demonstrated reproducible calcium and BOLD responses to the same stimuli. COMPARISON WITH EXISTING METHOD(S): Taken together, simultaneous GCaMP6-based fiber photometry and fMRI recording presents a novel, artifact-free approach to simultaneously measuring neural and fMRI signals. Furthermore, given the cell-type specificity of GCaMP6, this approach has the potential to mechanistically dissect the contributions of individual neuron populations to BOLD signal, and ultimately reveal its underlying neural mechanisms. CONCLUSIONS: The current study established the method for simultaneous GCaMP6-based fiber photometry and fMRI in rats.

Global reduction of information exchange during anesthetic-induced unconsciousness.

During anesthetic-induced unconsciousness (AIU), the brain undergoes a dramatic change in its capacity to exchange information between regions. However, the spatial distribution of information exchange loss/gain across the entire brain remains elusive. In the present study, we acquired and analyzed resting-state functional magnetic resonance imaging (rsfMRI) data in rats during wakefulness and graded levels of consciousness induced by incrementally increasing the concentration of isoflurane. We found that, regardless of spatial scale, the functional connectivity (FC) change (i.e., ∆FC) was proportionally dependent on the FC strength at the awake state across all connections. This dependency became stronger at higher doses of isoflurane. In addition, the relative FC change at each anesthetized condition (i.e., ∆FC normalized to the corresponding FC strength at the awake state) was exclusively negative across the whole brain, indicating a global loss of meaningful information exchange between brain regions during AIU. To further support this notion, we showed that during unconsciousness, the entropy of rsfMRI signal increased to a value comparable to random noise while the mutual information decreased appreciably. Importantly, consistent results were obtained when unconsciousness was induced by dexmedetomidine, an anesthetic agent with a distinct molecular action than isoflurane. This result indicates that the observed global reduction in information exchange may be agent invariant. Taken together, these findings provide compelling neuroimaging evidence suggesting that the brain undergoes a widespread disruption in the exchange of meaningful information during AIU and that this phenomenon may represent a common system-level neural mechanism of AIU.

Dynamic Connectivity Patterns in Conscious and Unconscious Brain.

Brain functional connectivity undergoes dynamic changes from the awake to unconscious states. However, how the dynamics of functional connectivity patterns are linked to consciousness at the behavioral level remains elusive. In this study, we acquired resting-state functional magnetic resonance imaging data during wakefulness and graded levels of consciousness in rats. Data were analyzed using a dynamic approach combining the sliding window method and k-means clustering. Our results demonstrate that whole-brain networks contained several quasi-stable patterns that dynamically recurred from the awake state into anesthetized states. Remarkably, two brain connectivity states with distinct spatial similarity to the structure of anatomical connectivity were strongly biased toward high and low consciousness levels, respectively. These results provide compelling neuroimaging evidence linking the dynamics of whole-brain functional connectivity patterns and states of consciousness at the behavioral level.

Time to wake up: Studying neurovascular coupling and brain-wide circuit function in the un-anesthetized animal.

Functional magnetic resonance imaging (fMRI) has allowed the noninvasive study of task-based and resting-state brain dynamics in humans by inferring neural activity from blood-oxygenation-level dependent (BOLD) signal changes. An accurate interpretation of the hemodynamic changes that underlie fMRI signals depends on the understanding of the quantitative relationship between changes in neural activity and changes in cerebral blood flow, oxygenation and volume. While there has been extensive study of neurovascular coupling in anesthetized animal models, anesthesia causes large disruptions of brain metabolism, neural responsiveness and cardiovascular function. Here, we review work showing that neurovascular coupling and brain circuit function in the awake animal are profoundly different from those in the anesthetized state. We argue that the time is right to study neurovascular coupling and brain circuit function in the awake animal to bridge the physiological mechanisms that underlie animal and human neuroimaging signals, and to interpret them in light of underlying neural mechanisms. Lastly, we discuss recent experimental innovations that have enabled the study of neurovascular coupling and brain-wide circuit function in un-anesthetized and behaving animal models.

Effect of the polydispersity of RBCs on the recovery rate of RBCs during the removal of CPAs.

In the process of removing cryoprotectants from cryopreserved blood, the theoretically optimal operating condition, which is based on the assumption that the distribution of red blood cells is uniform, is often used to reduce or even avoid the hypotonic damage to cells. However, due to the polydispersity of cells, the optimal condition is actually not reliable. In this study, based on the discrete concept developed in our previous work, the effect of the polydispersity on the recovery rate of cells in the dilution-filtration system was statistically investigated by assigning three random parameters, isotonic cell volume, cell surface area, and osmotically inactive cell volume, to cells in small units of blood. The results show that, due to the polydispersity, the real recovery rate deviates from the ideal value that is based on uniform distribution. The deviation significantly increases with the standard errors of cell parameters, and it can be also magnified by high cryoprotectant concentrations. Under the effect of polydispersity, the uniform distribution-based optimized blood or diluent flow rate is not perfect. In practice, one should adopt a more conservative blood or diluent flow rate so that the hypotonic damage to cells can be further reduced.

Statistical estimation of red blood cell osmotic damage during cryoprotective agent removal from cryopreserved blood.

Statistical estimation of the osmotic damage of red blood cells (RBCs) during the removal of cryoprotective agents (CPAs) from cryopreserved blood has been a very difficult issue. In this paper, the discrete mass transfer model developed in our previous work is modified to study the volume variation of individual RBCs and thereby to estimate the osmotic damage of all RBCs statistically during CPA removal by the dilution-concentration method we proposed recently. The model is validated with respect to the experimental results either with or without RBCs. Then, it is used to investigate the effects of blood volume, hematocrit, blood and diluent flow rates on the osmotic damage of RBCs, as well as the washing time of CPAs. Our results show that both the increase of blood flow rates and the decrease of diluent flow rates can bring about a reduction in osmotic damage of RBCs; however, only the former can cause a decrease in the washing time of CPAs. The blood volume could also affect the osmotic damage of RBCs. For a given flow condition, there could exist an optimal blood volume range for the dilution-concentration system. The effect of blood volume could be alleviated by an increase in the dilution region volume. In addition, the osmotic damage of RBCs decreases as the hematocrit decreases. Therefore, in practice, the increase of blood flow rates is the best solution to reduce both the osmotic damage of RBCs and the washing time of CPAs simultaneously. A lower hematocrit in the cryopreserved blood and/or longer tubing in the dilution region are also recommended to achieve better performance for the dilution-concentration method.