Brain-wide ongoing activity is responsible for significant cross-trial BOLD variability.

A notorious issue of task-based functional magnetic resonance imaging (fMRI) is its large cross-trial variability. To quantitatively characterize this variability, the blood oxygenation level-dependent (BOLD) signal can be modeled as a linear summation of a stimulation-relevant and an ongoing (i.e. stimulation-irrelevant) component. However, systematic investigation on the spatiotemporal features of the ongoing BOLD component and how these features affect the BOLD response is still lacking. Here we measured fMRI responses to light onsets and light offsets in awake rats. The neuronal response was simultaneously recorded with calcium-based fiber photometry. We established that between-region BOLD signals were highly correlated brain-wide at zero time lag, including regions that did not respond to visual stimulation, suggesting that the ongoing activity co-fluctuates across the brain. Removing this ongoing activity reduced cross-trial variability of the BOLD response by ~30% and increased its coherence with the Ca2+ signal. Additionally, the negative ongoing BOLD activity sometimes dominated over the stimulation-driven response and contributed to the post-stimulation BOLD undershoot. These results suggest that brain-wide ongoing activity is responsible for significant cross-trial BOLD variability, and this component can be reliably quantified and removed to improve the reliability of fMRI response. Importantly, this method can be generalized to virtually all fMRI experiments without changing stimulation paradigms.

Gaining insight into the neural basis of resting-state fMRI signal.

The blood oxygenation level-dependent (BOLD)-based resting-state functional magnetic resonance imaging (rsfMRI) has been widely used as a non-invasive tool to map brain-wide connectivity architecture. However, the neural basis underpinning the resting-state BOLD signal remains elusive. In this study, we combined simultaneous calcium-based fiber photometry with rsfMRI in awake animals to examine the relationship of the BOLD signal and spiking activity at the resting state. We observed robust couplings between calcium and BOLD signals in the dorsal hippocampus as well as other distributed areas in the default mode network (DMN), suggesting that the calcium measurement can reliably predict the rsfMRI signal. In addition, using the calcium signal recorded as the ground truth, we assessed the impacts of different rsfMRI data preprocessing pipelines on functional connectivity mapping. Overall, our results provide important evidence suggesting that spiking activity measured by the calcium signal plays a key role in the neural mechanism of resting-state BOLD signal.

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.

Brain network reorganization after targeted attack at a hub region.

The architecture of brain networks has been extensively studied in multiple species. However, exactly how the brain network reconfigures when a local region, particularly a hub region, stops functioning remains elusive. By combining chemogenetics and resting-state functional magnetic resonance imaging (rsfMRI) in an awake rodent model, we investigated the causal impact of acutely inactivating a hub region (i.e. the dorsal anterior cingulate cortex) on brain network properties. We found that suppressing neural activity in a hub could have a ripple effect that went beyond the hub-related connections and propagated to other neural connections across multiple brain systems. In addition, hub dysfunction affected the topological architecture of the whole-brain network in terms of the network resilience and segregation. Selectively inhibiting excitatory neurons in the hub further changed network integration. None of these changes were observed in sham rats or when a non-hub region (i.e. the primary visual cortex) was perturbed. This study has established a system that allows for mechanistically dissecting the relationship between local regions and brain network properties. Our data provide direct evidence supporting the hypothesis that acute dysfunction of a brain hub can cause large-scale network changes. These results also provide a comprehensive framework documenting the differential impact of hub versus non-hub nodes on network dynamics.

Increased wiring cost during development is driven by long-range cortical, but not subcortical connections.

The brain undergoes a protracted, metabolically expensive maturation process from childhood to adulthood. Therefore, it is crucial to understand how network cost is distributed among different brain systems as the brain matures. To address this issue, here we examined developmental changes in wiring cost and brain network topology using resting-state functional magnetic resonance imaging (rsfMRI) data longitudinally collected in awake rats from the juvenile age to adulthood. We found that the wiring cost increased in the vast majority of cortical connections but decreased in most subcortico-subcortical connections. Importantly, the developmental increase in wiring cost was dominantly driven by long-range cortical, but not subcortical connections, which was consistent with more pronounced increase in network integration in the cortical network. These results collectively indicate that there is a non-uniform distribution of network cost as the brain matures, and network resource is dominantly consumed for the development of the cortex, but not subcortex from the juvenile age to adulthood.

An open database of resting-state fMRI in awake rats.

Rodent models are essential to translational research in health and disease. Investigation in rodent brain function and organization at the systems level using resting-state functional magnetic resonance imaging (rsfMRI) has become increasingly popular. Due to this rapid progress, publicly shared rodent rsfMRI databases can be of particular interest and importance to the scientific community, as inspired by human neuroscience and psychiatric research that are substantially facilitated by open human neuroimaging datasets. However, such databases in rats are still rare. In this paper, we share an open rsfMRI database acquired in 90 rats with a well-established awake imaging paradigm that avoids anesthesia interference. Both raw and preprocessed data are made publicly available. Procedures in data preprocessing to remove artefacts induced by the scanner, head motion and non-neural physiological noise are described in details. We also showcase inter-regional functional connectivity and functional networks obtained from the database.

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.

Functional atlas of the awake rat brain: A neuroimaging study of rat brain specialization and integration.

Connectivity-based parcellation approaches present an innovative method to segregate the brain into functionally specialized regions. These approaches have significantly advanced our understanding of the human brain organization. However, parallel progress in animal research is sparse. Using resting-state fMRI data and a novel, data-driven parcellation method, we have obtained robust functional parcellations of the rat brain. These functional parcellations reveal the regional specialization of the rat brain, which exhibited high within-parcel homogeneity and high reproducibility across animals. Graph analysis of the whole-brain network constructed based on these functional parcels indicates that the rat brain has a topological organization similar to humans, characterized by both segregation and integration. Our study also provides compelling evidence that the cingulate cortex is a functional hub region conserved from rodents to humans. Together, this study has characterized the rat brain specialization and integration, and has significantly advanced our understanding of the rat brain organization. In addition, it is valuable for studies of comparative functional neuroanatomy in mammalian brains.

Neuropharmacology Involvement of dopamine D3 receptor in impulsive choice decision-making in male rats.

Impulsive decision-making has been linked to impulse control disorders and substance use disorders. However, the neural mechanisms underlying impulsive choice are not fully understood. While previous PET imaging and autoradiography studies have shown involvement of dopamine and D2/3 receptors in impulsive behavior, the roles of distinct D1, D2, and D3 receptors in impulsive decision-making remain unclear. In this study, we used a food reward delay-discounting task (DDT) to identify low- and high-impulsive rats, in which low-impulsive rats exhibited preference for large delayed reward over small immediate rewards, while high-impulsive rats showed the opposite preference. We then examined D1, D2, and D3 receptor gene expression using RNAscope in situ hybridization assays. We found that high-impulsive male rats exhibited lower levels of D2 and D3, and particularly D3, receptor expression in the nucleus accumbens (NAc), with no significant changes in the insular, prelimbic, and infralimbic cortices. Based on these findings, we further explored the role of the D3 receptor in impulsive decision-making. Systemic administration of a selective D3 receptor agonist (FOB02-04) significantly reduced impulsive choices in high-impulsive rats but had no effects in low-impulsive rats. Conversely, a selective D3 receptor antagonist (VK4-116) produced increased both impulsive and omission choices in both groups of rats. These findings suggest that impulsive decision-making is associated with a reduction in D3 receptor expression in the NAc. Selective D3 receptor agonists, but not antagonists, may hold therapeutic potentials for mitigating impulsivity in high-impulsive subjects.

Biomarkers of Parkinson's Disease: From Basic Research to Clinical Practice.

Parkinson's disease (PD) is a common neurodegenerative disease characterized pathologically by dopaminergic neuron loss and the formation of Lewy bodies, which are enriched with aggregated α-synuclein (α-syn). PD currently has no cure, but therapeutic strategies are available to alleviate symptoms. Early diagnosis can greatly improve therapeutic interventions, but the clinical diagnosis of PD remains challenging and depends mainly on clinical features and imaging tests. Efficient and specific biomarkers are crucial for the diagnosis, monitoring, and evaluation of PD. Here, we reviewed the biomarkers of PD in different tissues and biofluids, along with the current clinical biochemical detection methods. We found that the sensitivity and specificity of single biomarkers are limited, and selecting appropriate indicators for combined detection can improve the diagnostic accuracy of PD.

In Silico Analyses, Experimental Verification and Application in DNA Vaccines of Ebolavirus GP-Derived pan-MHC-II-Restricted Epitopes.

(1) Background and Purpose: Ebola virus (EBOV) is the causative agent of Ebola virus disease (EVD), which causes extremely high mortality and widespread epidemics. The only glycoprotein (GP) on the surface of EBOV particles is the key to mediating viral invasion into host cells. DNA vaccines for EBOV are in development, but their effectiveness is unclear. The lack of immune characteristics resides in antigenic MHC class II reactivity. (2) Methods: We selected MHC-II molecules from four human leukocyte antigen II (HLA-II) superfamilies with 98% population coverage and eight mouse H2-I alleles. IEDB, NetMHCIIpan, SYFPEITHI, and Rankpep were used to screen MHC-II-restricted epitopes with high affinity for EBOV GP. Further immunogenicity and conservation analyses were performed using VaxiJen and BLASTp, respectively. EpiDock was used to simulate molecular docking. Cluster analysis and binding affinity analysis of EBOV GP epitopes and selected MHC-II molecules were performed using data from NetMHCIIpan. The selective GP epitopes were verified by the enzyme-linked immunospot (ELISpot) assay using splenocytes of BALB/c (H2d), C3H, and C57 mice after DNA vaccine pVAX-GPEBO immunization. Subsequently, BALB/c mice were immunized with Protein-GPEBO, plasmid pVAX-GPEBO, and pVAX-LAMP/GPEBO, which encoded EBOV GP. The dominant epitopes of BALB/c (H-2-I-AdEd genotype) mice were verified by the enzyme-linked immunospot (ELISpot) assay. It is also used to evaluate and explore the advantages of pVAX-LAMP/GPEBO and the reasons behind them. (3) Results: Thirty-one HLA-II-restricted and 68 H2-I-restricted selective epitopes were confirmed to have high affinity, immunogenicity, and conservation. Nineteen selective epitopes have cross-species reactivity with good performance in MHC-II molecular docking. The ELISpot results showed that pVAX-GPEBO could induce a cellular immune response to the synthesized selective peptides. The better immunoprotection of the DNA vaccines pVAX-LAMP/GPEBO coincides with the enhancement of the MHC class II response. (4) Conclusions: Promising MHC-II-restricted candidate epitopes of EBOV GP were identified in humans and mice, which is of great significance for the development and evaluation of Ebola vaccines.

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.

Lack of Causal Roles of Cannabinoid and Dopamine Neurotransmitter Systems in Orbitofrontal and Piriform Cortex in Fentanyl Relapse in Rats.

The orbitofrontal cortex (OFC) and piriform cortex (Pir) play a role in fentanyl relapse after food choice-induced voluntary abstinence, a procedure mimicking abstinence because of availability of alternative nondrug rewards. We used in situ hybridization and pharmacology to determine the role of OFC and Pir cannabinoid and dopamine receptors in fentanyl relapse. We trained male and female rats to self-administer food pellets for 6 d (6 h/d) and intravenous fentanyl (2.5 µg/kg/infusion) for 12 d (6 h/d). We assessed fentanyl relapse after 12 discrete choice sessions between fentanyl and food (20 trials/d), in which rats voluntarily reduced fentanyl self-administration. We used RNAscope to determine whether fentanyl relapse is associated with activity (indicated by Fos) in OFC and Pir cells expressing Cnr1 [which encodes cannabinoid 1 (CB1) receptors] or Drd1 and Drd2 (which encode dopamine D1 and D2 receptors). We injected a CB1 receptor antagonist or agonist (0.3 or 1.0 µg AM251 or WIN55,212-2/hemisphere) into OFC or a dopamine D1 receptor antagonist (1.0 or 3.0 µg SCH39166/hemisphere) into Pir to determine the effect on fentanyl relapse. Fentanyl relapse was associated with OFC cells co-expressing Fos and Cnr1 and Pir cells co-expressing Fos and Drd1 However, injections of the CB1 receptor antagonist AM251 or agonist WIN55,212-2 into OFC or the dopamine D1 receptor antagonist SCH39166 into Pir had no effect on fentanyl relapse. Fentanyl relapse is associated with activation of Cnr1-expressing OFC cells and Drd1-expressing Pir cells, but pharmacological manipulations do not support causal roles of OFC CB1 receptors or Pir dopamine D1 receptors in fentanyl relapse.

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.