Space wandering in the rodent default mode network.

The default mode network (DMN) is a large-scale brain network known to be suppressed during a wide range of cognitive tasks. However, our comprehension of its role in naturalistic and unconstrained behaviors has remained elusive because most research on the DMN has been conducted within the restrictive confines of MRI scanners. Here, we use multisite GCaMP (a genetically encoded calcium indicator) fiber photometry with simultaneous videography to probe DMN function in awake, freely exploring rats. We examined neural dynamics in three core DMN nodes-the retrosplenial cortex, cingulate cortex, and prelimbic cortex-as well as the anterior insula node of the salience network, and their association with the rats' spatial exploration behaviors. We found that DMN nodes displayed a hierarchical functional organization during spatial exploration, characterized by stronger coupling with each other than with the anterior insula. Crucially, these DMN nodes encoded the kinematics of spatial exploration, including linear and angular velocity. Additionally, we identified latent brain states that encoded distinct patterns of time-varying exploration behaviors and found that higher linear velocity was associated with enhanced DMN activity, heightened synchronization among DMN nodes, and increased anticorrelation between the DMN and anterior insula. Our findings highlight the involvement of the DMN in collectively and dynamically encoding spatial exploration in a real-world setting. Our findings challenge the notion that the DMN is primarily a "task-negative" network disengaged from the external world. By illuminating the DMN's role in naturalistic behaviors, our study underscores the importance of investigating brain network function in ecologically valid contexts.

Optogenetic fMRI reveals therapeutic circuits of subthalamic nucleus deep brain stimulation.

While deep brain stimulation (DBS) is widely employed for managing motor symptoms in Parkinson's disease (PD), its exact circuit mechanisms remain controversial. To identify the neural targets affected by therapeutic DBS in PD, we analyzed DBS-evoked whole brain activity in female hemi-parkinsonian rats using function magnetic resonance imaging (fMRI). We delivered subthalamic nucleus (STN) DBS at various stimulation pulse repetition rates using optogenetics, allowing unbiased examinations of cell-type specific STN feed-forward neural activity. Unilateral STN optogenetic stimulation elicited pulse repetition rate-dependent alterations of blood-oxygenation-level-dependent (BOLD) signals in SNr (substantia nigra pars reticulata), GP (globus pallidus), and CPu (caudate putamen). Notably, these manipulations effectively ameliorated pathological circling behavior in animals expressing the kinetically faster Chronos opsin, but not in animals expressing ChR2. Furthermore, mediation analysis revealed that the pulse repetition rate-dependent behavioral rescue was significantly mediated by optogenetically induced activity changes in GP and CPu, but not in SNr. This suggests that the activation of GP and CPu are critically involved in the therapeutic mechanisms of STN DBS.

Distinct neurochemical influences on fMRI response polarity in the striatum.

The striatum, known as the input nucleus of the basal ganglia, is extensively studied for its diverse behavioral roles. However, the relationship between its neuronal and vascular activity, vital for interpreting functional magnetic resonance imaging (fMRI) signals, has not received comprehensive examination within the striatum. Here, we demonstrate that optogenetic stimulation of dorsal striatal neurons or their afferents from various cortical and subcortical regions induces negative striatal fMRI responses in rats, manifesting as vasoconstriction. These responses occur even with heightened striatal neuronal activity, confirmed by electrophysiology and fiber-photometry. In parallel, midbrain dopaminergic neuron optogenetic modulation, coupled with electrochemical measurements, establishes a link between striatal vasodilation and dopamine release. Intriguingly, in vivo intra-striatal pharmacological manipulations during optogenetic stimulation highlight a critical role of opioidergic signaling in generating striatal vasoconstriction. This observation is substantiated by detecting striatal vasoconstriction in brain slices after synthetic opioid application. In humans, manipulations aimed at increasing striatal neuronal activity likewise elicit negative striatal fMRI responses. Our results emphasize the necessity of considering vasoactive neurotransmission alongside neuronal activity when interpreting fMRI signal.

Intrinsic Functional Connectivity between the Anterior Insular and Retrosplenial Cortex as a Moderator and Consequence of Cocaine Self-Administration in Rats.

While functional brain imaging studies in humans suggest that chronic cocaine use alters functional connectivity (FC) within and between key large-scale brain networks, including the default mode network (DMN), the salience network (SN), and the central executive network (CEN), cross-sectional studies in humans are challenging to obtain brain FC prior to cocaine use. Such information is critical to reveal the relationship between individual's brain FC and the subsequent development of cocaine dependence and brain changes during abstinence. Here, we performed a longitudinal study examining functional magnetic resonance imaging (fMRI) data in male rats (n = 7), acquired before cocaine self-administration (baseline), on 1 d of abstinence following 10 d of cocaine self-administration, and again after 30 d of experimenter-imposed abstinence. Using repeated-measures analysis of variance (ANOVA) with network-based statistics (NBS), significant connectivity changes were found between anterior insular cortex (AI) of the SN, retrosplenial cortex (RSC) of the DMN, somatosensory cortex, and caudate-putamen (CPu), with AI-RSC FC showing the most robust changes between baseline and 1 d of abstinence. Additionally, the level of escalated cocaine intake is associated with AI-RSC and AI-CPu FC changes between 1 d and 30 d of abstinence; further, the subjects' AI-RSC FC prior to cocaine intake is a significant moderator for the AI-RSC changes during abstinence. These results provide novel insights into the roles of AI-RSC FC before and after cocaine intake and suggest this circuit to be a potential target to modulate large-scale network and associated behavioral changes in cocaine use disorders.

Complement receptor 3-mediated neurotoxic glial activation contributes to rotenone-induced cognitive decline in mice.

Microglia-mediated chronic neuroinflammation has been associated with cognitive decline induced by rotenone, a well-known neurotoxic pesticide used in agriculture. However, the mechanisms remain unclear. This work aimed to elucidate the role of complement receptor 3 (CR3), a highly expressed receptor in microglia, in cognitive deficits induced by rotenone. Rotenone up-regulated the expression of CR3 in the hippocampus and cortex area of mice. CR3 deficiency markedly ameliorated rotenone-induced cognitive impairments, neurodegeneration and phosphorylation (Ser129) of α-synuclein in mice. CR3 deficiency also attenuated rotenone-stimulated microglial M1 activation. In microglial cells, siRNA-mediated knockdown of CR3 impeded, while CR3 activation induced by LL-37 exacerbated, rotenone-induced microglial M1 activation. Mechanistically, CR3 deficiency blocked rotenone-induced activation of nuclear factor κB (NF-κB), signal transducer and activator of transcription 1 (STAT1) and STAT3 signaling pathways. Pharmacological inhibition of NF-κB or STAT3 but not STAT1 was confirmed to suppress microglial M1 activation elicited by rotenone. Further study revealed that CR3 deficiency or knockdown also reduced rotenone-induced expression of C3, an A1 astrocyte marker, and production of microglial C1q, TNFα and IL-1α, a cocktail for activated microglia to induce neurotoxic A1 astrocytes, via NF-κB and STAT3 pathways. Finally, a small molecule modulator of CR3 efficiently mitigated rotenone-elicited cognitive deficits in mice even administered after the establishment of cognitive dysfunction. Taken together, our findings demonstrated that CR3 is a key factor in mediating neurotoxic glial activation and subsequent cognitive impairments in rotenone-treated mice, giving novel insights into the immunopathogenesis of cognitive impairments in pesticide-related Parkinsonism.

Space wandering in the rodent default mode network.

The default mode network (DMN) is a large-scale brain network known to be suppressed during a wide range of cognitive tasks. However, our comprehension of its role in naturalistic and unconstrained behaviors has remained elusive because most research on the DMN has been conducted within the restrictive confines of MRI scanners. Here we use multisite GCaMP fiber photometry with simultaneous videography to probe DMN function in awake, freely exploring rats. We examined neural dynamics in three core DMN nodes- the retrosplenial cortex, cingulate cortex, and prelimbic cortex- as well as the anterior insula node of the salience network, and their association with the rats' spatial exploration behaviors. We found that DMN nodes displayed a hierarchical functional organization during spatial exploration, characterized by stronger coupling with each other than with the anterior insula. Crucially, these DMN nodes encoded the kinematics of spatial exploration, including linear and angular velocity. Additionally, we identified latent brain states that encoded distinct patterns of time-varying exploration behaviors and discovered that higher linear velocity was associated with enhanced DMN activity, heightened synchronization among DMN nodes, and increased anticorrelation between the DMN and anterior insula. Our findings highlight the involvement of the DMN in collectively and dynamically encoding spatial exploration in a real-world setting. Our findings challenge the notion that the DMN is primarily a "task-negative" network disengaged from the external world. By illuminating the DMN's role in naturalistic behaviors, our study underscores the importance of investigating brain network function in ecologically valid contexts.

Acute alcohol induces greater dose-dependent increase in the lateral cortical network functional connectivity in adult than adolescent rats.

Alcohol misuse and, particularly adolescent drinking, is a major public health concern. While evidence suggests that adolescent alcohol use affects frontal brain regions that are important for cognitive control over behavior little is known about how acute alcohol exposure alters large-scale brain networks and how sex and age may moderate such effects. Here, we employ a recently developed functional magnetic resonance imaging (fMRI) protocol to acquire rat brain functional connectivity data and use an established analytical pipeline to examine the effect of sex, age, and alcohol dose on connectivity within and between three major rodent brain networks: defaul mode, salience, and lateral cortical network. We identify the intra- and inter-network connectivity differences and establish moderation models to reveal significant influences of age on acute alcohol-induced lateral cortical network connectivity. Through this work, we make brain-wide isotropic fMRI data with acute alcohol challenge publicly available, with the hope to facilitate future discovery of brain regions/circuits that are causally relevant to the impact of acute alcohol use.

Compensatory remodeling of a septo-hippocampal GABAergic network in the triple transgenic Alzheimer's mouse model.

BACKGROUND: Alzheimer's disease (AD) is characterized by a progressive loss of memory that cannot be efficiently managed by currently available AD therapeutics. So far, most treatments for AD that have the potential to improve memory target neural circuits to protect their integrity. However, the vulnerable neural circuits and their dynamic remodeling during AD progression remain largely undefined. METHODS: Circuit-based approaches, including anterograde and retrograde tracing, slice electrophysiology, and fiber photometry, were used to investigate the dynamic structural and functional remodeling of a GABAergic circuit projected from the medial septum (MS) to the dentate gyrus (DG) in 3xTg-AD mice during AD progression. RESULTS: We identified a long-distance GABAergic circuit that couples highly connected MS and DG GABAergic neurons during spatial memory encoding. Furthermore, we found hyperactivity of DG interneurons during early AD, which persisted into late AD stages. Interestingly, MS GABAergic projections developed a series of adaptive strategies to combat DG interneuron hyperactivity. During early-stage AD, MS-DG GABAergic projections exhibit increased inhibitory synaptic strength onto DG interneurons to inhibit their activities. During late-stage AD, MS-DG GABAergic projections form higher anatomical connectivity with DG interneurons and exhibit aberrant outgrowth to increase the inhibition onto DG interneurons. CONCLUSION: We report the structural and functional remodeling of the MS-DG GABAergic circuit during disease progression in 3xTg-AD mice. Dynamic MS-DG GABAergic circuit remodeling represents a compensatory mechanism to combat DG interneuron hyperactivity induced by reduced GABA transmission.

Optogenetic stimulation of anterior insular cortex neurons in male rats reveals causal mechanisms underlying suppression of the default mode network by the salience network.

The salience network (SN) and default mode network (DMN) play a crucial role in cognitive function. The SN, anchored in the anterior insular cortex (AI), has been hypothesized to modulate DMN activity during stimulus-driven cognition. However, the causal neural mechanisms underlying changes in DMN activity and its functional connectivity with the SN are poorly understood. Here we combine feedforward optogenetic stimulation with fMRI and computational modeling to dissect the causal role of AI neurons in dynamic functional interactions between SN and DMN nodes in the male rat brain. Optogenetic stimulation of Chronos-expressing AI neurons suppressed DMN activity, and decreased AI-DMN and intra-DMN functional connectivity. Our findings demonstrate that feedforward optogenetic stimulation of AI neurons induces dynamic suppression and decoupling of the DMN and elucidates previously unknown features of rodent brain network organization. Our study advances foundational knowledge of causal mechanisms underlying dynamic cross-network interactions and brain network switching.

Neuronal dynamics of the default mode network and anterior insular cortex: Intrinsic properties and modulation by salient stimuli.

The default mode network (DMN) is critical for self-referential mental processes, and its dysfunction is implicated in many neuropsychiatric disorders. However, the neurophysiological properties and task-based functional organization of the rodent DMN are poorly understood, limiting its translational utility. Here, we combine fiber photometry with functional magnetic resonance imaging (fMRI) and computational modeling to characterize dynamics of putative rat DMN nodes and their interactions with the anterior insular cortex (AI) of the salience network. Our analysis revealed neuronal activity changes in AI and DMN nodes preceding fMRI-derived DMN activations and cyclical transitions between brain network states. Furthermore, we demonstrate that salient oddball stimuli suppress the DMN and enhance AI neuronal activity and that the AI causally inhibits the retrosplenial cortex, a prominent DMN node. These findings elucidate the neurophysiological foundations of the rodent DMN, its spatiotemporal dynamical properties, and modulation by salient stimuli, paving the way for future translational studies.

Dysfunction of the noradrenergic system drives inflammation, α-synucleinopathy, and neuronal loss in mouse colon.

Clinical and pathological evidence revealed that α-synuclein (α-syn) pathology seen in PD patients starts in the gut and spreads via anatomically connected structures from the gut to the brain. Our previous study demonstrated that depletion of central norepinephrine (NE) disrupted brain immune homeostasis, producing a spatiotemporal order of neurodegeneration in the mouse brain. The purpose of this study was 1) to determine the role of peripheral noradrenergic system in the maintenance of gut immune homeostasis and in the pathogenesis of PD and 2) to investigate whether NE-depletion induced PD-like α-syn pathological changes starts from the gut. For these purposes, we investigated time-dependent changes of α-synucleinopathy and neuronal loss in the gut following a single injection of DSP-4 (a selective noradrenergic neurotoxin) to A53T-SNCA (human mutant α-syn) over-expression mice. We found DPS-4 significantly reduced the tissue level of NE and increased immune activities in gut, characterized by increased number of phagocytes and proinflammatory gene expression. Furthermore, a rapid-onset of α-syn pathology was observed in enteric neurons after 2 weeks and delayed dopaminergic neurodegeneration in the substantia nigra was detected after 3-5 months, associated with the appearance of constipation and impaired motor function, respectively. The increased α-syn pathology was only observed in large, but not in the small, intestine, which is similar to what was observed in PD patients. Mechanistic studies reveal that DSP-4-elicited upregulation of NADPH oxidase (NOX2) initially occurred only in immune cells during the acute intestinal inflammation stage, and then spread to enteric neurons and mucosal epithelial cells during the chronic inflammation stage. The upregulation of neuronal NOX2 correlated well with the extent of α-syn aggregation and subsequent enteric neuronal loss, suggesting that NOX2-generated reactive oxygen species play a key role in α-synucleinopathy. Moreover, inhibiting NOX2 by diphenyleneiodonium or restoring NE function by salmeterol (a ß2-receptor agonist) significantly attenuated colon inflammation, α-syn aggregation/propagation, and enteric neurodegeneration in the colon and ameliorated subsequent behavioral deficits. Taken together, our model of PD shows a progressive pattern of pathological changes from the gut to the brain and suggests a potential role of the noradrenergic dysfunction in the pathogenesis of PD.

Putative neurochemical and cell type contributions to hemodynamic activity in the rodent caudate putamen.

Functional magnetic resonance imaging (fMRI) is widely used by researchers to noninvasively monitor brain-wide activity. The traditional assumption of a uniform relationship between neuronal and hemodynamic activity throughout the brain has been increasingly challenged. This relationship is now believed to be impacted by heterogeneously distributed cell types and neurochemical signaling. To date, most cell-type- and neurotransmitter-specific influences on hemodynamics have been examined within the cortex and hippocampus of rodent models, where glutamatergic signaling is prominent. However, neurochemical influences on hemodynamics are relatively unknown in largely GABAergic brain regions such as the rodent caudate putamen (CPu). Given the extensive contribution of CPu function and dysfunction to behavior, and the increasing focus on this region in fMRI studies, improved understanding of CPu hemodynamics could have broad impacts. Here we discuss existing findings on neurochemical contributions to hemodynamics as they may relate to the CPu with special consideration for how these contributions could originate from various cell types and circuits. We hope this review can help inform the direction of future studies as well as interpretation of fMRI findings in the CPu.

Translational landscape of direct cardiac reprogramming reveals a role of Ybx1 in repressing cardiac fate acquisition.

Direct reprogramming of fibroblasts into induced cardiomyocytes holds great promise for heart regeneration. Although considerable progress has been made in understanding the transcriptional and epigenetic mechanisms of iCM reprogramming, its translational regulation remains largely unexplored. Here, we characterized the translational landscape of iCM reprogramming through integrative ribosome and transcriptomic profiling, and found extensive translatome repatterning during this process. Loss of function screening for translational regulators uncovered Ybx1 as a critical barrier to iCM induction. In a mouse model of myocardial infarction, removing Ybx1 enhanced in vivo reprogramming, resulting in improved heart function and reduced scar size. Mechanistically, Ybx1 depletion de-repressed the translation of its direct targets SRF and Baf60c, both of which mediated the effect of Ybx1 depletion on iCM generation. Furthermore, removal of Ybx1 allowed single factor Tbx5-mediated iCM conversion. In summary, this study revealed a new layer of regulatory mechanism that controls cardiac reprogramming at the translational level.

Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy.

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of structural changes caused by genetic or environmental perturbations. Whole-brain imaging results in more accurate quantification of cells and the study of region-specific differences that may be missed with commonly used microscopy of physically sectioned tissue. Using light-sheet microscopy to image cleared brains greatly increases acquisition speed as compared to confocal microscopy. Although these images produce very large amounts of brain structural data, most computational tools that perform feature quantification in images of cleared tissue are limited to counting sparse cell populations, rather than all nuclei. Here, we demonstrate NuMorph (Nuclear-Based Morphometry), a group of analysis tools, to quantify all nuclei and nuclear markers within annotated regions of a postnatal day 4 (P4) mouse brain after clearing and imaging on a light-sheet microscope. We describe magnetic resonance imaging (MRI) to measure brain volume prior to shrinkage caused by tissue clearing dehydration steps, tissue clearing using the iDISCO+ method, including immunolabeling, followed by light-sheet microscopy using a commercially available platform to image mouse brains at cellular resolution. We then demonstrate this image analysis pipeline using NuMorph, which is used to correct intensity differences, stitch image tiles, align multiple channels, count nuclei, and annotate brain regions through registration to publicly available atlases. We designed this approach using publicly available protocols and software, allowing any researcher with the necessary microscope and computational resources to perform these techniques. These tissue clearing, imaging, and computational tools allow measurement and quantification of the three-dimensional (3D) organization of cell-types in the cortex and should be widely applicable to any wild-type/knockout mouse study design.

Spectral fiber photometry derives hemoglobin concentration changes for accurate measurement of fluorescent sensor activity.

Fiber photometry is an emerging technique for recording fluorescent sensor activity in the brain. However, significant hemoglobin absorption artifacts in fiber photometry data may be misinterpreted as sensor activity changes. Because hemoglobin exists widely in the brain, and its concentration varies temporally, such artifacts could impede the accuracy of photometry recordings. Here we present use of spectral photometry and computational methods to quantify photon absorption effects by using activity-independent fluorescence signals, which can be used to derive oxy- and deoxy-hemoglobin concentration changes. Although these changes are often temporally delayed compared with the fast-responding fluorescence spikes, we found that erroneous interpretation may occur when examining pharmacology-induced sustained changes and that sometimes hemoglobin absorption could flip the GCaMP signal polarity. We provide hemoglobin-based correction methods to restore fluorescence signals and compare our results with other commonly used approaches. We also demonstrated the utility of spectral fiber photometry for delineating regional differences in hemodynamic response functions.

Simultaneous recording of neuronal and vascular activity in the rodent brain using fiber-photometry.

Coupling of hemodynamic responses to neuronal activity is the foundation of several functional neuroimaging techniques. Here, we provide three fiber-photometry approaches to simultaneously measure neuronal and vascular signals in the rodent brain using a spectrometer-based system. Two out of these three approaches allow the removal of hemoglobin (Hb)-absorption artifacts and restore the underlying neuronal activity. This technique is applicable to different fluorescent sensors and provides a more accurate measurement of hemodynamic response function in any location of the rodent brain. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022).

Chemogenetic stimulation of tonic locus coeruleus activity strengthens the default mode network.

The default mode network (DMN) of the brain is functionally associated with a wide range of behaviors. In this study, we used functional magnetic resonance imaging (fMRI), positron emission tomography (PET), and spectral fiber photometry to investigate the selective neuromodulatory effect of norepinephrine (NE)-releasing noradrenergic neurons in the locus coeruleus (LC) on the mouse DMN. Chemogenetic-induced tonic LC activity decreased cerebral blood volume (CBV) and glucose uptake and increased synchronous low-frequency fMRI activity within the frontal cortices of the DMN. Fiber photometry results corroborated these findings, showing that LC-NE activation induced NE release, enhanced calcium-weighted neuronal spiking, and reduced CBV in the anterior cingulate cortex. These data suggest that LC-NE alters conventional coupling between neuronal activity and CBV in the frontal DMN. We also demonstrated that chemogenetic activation of LC-NE neurons strengthened functional connectivity within the frontal DMN, and this effect was causally mediated by reduced modulatory inputs from retrosplenial and hippocampal regions to the association cortices of the DMN.

Altered Cortico-Subcortical Network After Adolescent Alcohol Exposure Mediates Behavioral Deficits in Flexible Decision-Making.

Behavioral flexibility, the ability to modify behavior according to changing conditions, is essential to optimize decision-making. Deficits in behavioral flexibility that persist into adulthood are one consequence of adolescent alcohol exposure, and another is decreased functional connectivity in brain structures involved in decision-making; however, a link between these two outcomes has not been established. We assessed effects of adolescent alcohol and sex on both Pavlovian and instrumental behaviors and resting-state functional connectivity MRI in adult animals to determine associations between behavioral flexibility and resting-state functional connectivity. Alcohol exposure impaired attentional set reversals and decreased functional connectivity among cortical and subcortical regions-of-interest that underlie flexible behavior. Moreover, mediation analyses indicated that adolescent alcohol-induced reductions in functional connectivity within a subnetwork of affected brain regions statistically mediated errors committed during reversal learning. These results provide a novel link between persistent reductions in brain functional connectivity and deficits in behavioral flexibility resulting from adolescent alcohol exposure.

Simultaneous fMRI and fast-scan cyclic voltammetry bridges evoked oxygen and neurotransmitter dynamics across spatiotemporal scales.

The vascular contributions of neurotransmitters to the hemodynamic response are gaining more attention in neuroimaging studies, as many neurotransmitters are vasomodulatory. To date, well-established electrochemical techniques that detect neurotransmission in high magnetic field environments are limited. Here, we propose an experimental setting enabling simultaneous fast-scan cyclic voltammetry (FSCV) and blood oxygenation level-dependent functional magnetic imaging (BOLD fMRI) to measure both local tissue oxygen and dopamine responses, and global BOLD changes, respectively. By using MR-compatible materials and the proposed data acquisition schemes, FSCV detected physiological analyte concentrations with high temporal resolution and spatial specificity inside of a 9.4 T MRI bore. We found that tissue oxygen and BOLD correlate strongly, and brain regions that encode dopamine amplitude differences can be identified via modeling simultaneously acquired dopamine FSCV and BOLD fMRI time-courses. This technique provides complementary neurochemical and hemodynamic information and expands the scope of studying the influence of local neurotransmitter release over the entire brain.

An isotropic EPI database and analytical pipelines for rat brain resting-state fMRI.

Resting-state functional magnetic resonance imaging (fMRI) has drastically expanded the scope of brain research by advancing our knowledge about the topologies, dynamics, and interspecies translatability of functional brain networks. Several databases have been developed and shared in accordance with recent key initiatives in the rodent fMRI community to enhance the transparency, reproducibility, and interpretability of data acquired at various sites. Despite these pioneering efforts, one notable challenge preventing efficient standardization in the field is the customary choice of anisotropic echo planar imaging (EPI) schemes with limited spatial coverage. Imaging with anisotropic resolution and/or reduced brain coverage has significant shortcomings including reduced registration accuracy and increased deviation in brain feature detection. Here we proposed a high-spatial-resolution (0.4 mm), isotropic, whole-brain EPI protocol for the rat brain using a horizontal slicing scheme that can maintain a functionally relevant repetition time (TR), avoid high gradient duty cycles, and offer unequivocal whole-brain coverage. Using this protocol, we acquired resting-state EPI fMRI data from 87 healthy rats under the widely used dexmedetomidine sedation supplemented with low-dose isoflurane on a 9.4 T MRI system. We developed an EPI template that closely approximates the Paxinos and Watson's rat brain coordinate system and demonstrated its ability to improve the accuracy of group-level approaches and streamline fMRI data pre-processing. Using this database, we employed a multi-scale dictionary-learning approach to identify reliable spatiotemporal features representing rat brain intrinsic activity. Subsequently, we performed k-means clustering on those features to obtain spatially discrete, functional regions of interest (ROIs). Using Euclidean-based hierarchical clustering and modularity-based partitioning, we identified the topological organizations of the rat brain. Additionally, the identified group-level FC network appeared robust across strains and sexes. The "triple-network" commonly adapted in human fMRI were resembled in the rat brain. Through this work, we disseminate raw and pre-processed isotropic EPI data, a rat brain EPI template, as well as identified functional ROIs and networks in standardized rat brain coordinates. We also make our analytical pipelines and scripts publicly available, with the hope of facilitating rat brain resting-state fMRI study standardization.