Resting State Brain Networks under Inverse Agonist versus Complete Knockout of the Cannabinoid Receptor 1.

The cannabinoid receptor 1 (CB1) is famous as the target of Δ9-tetrahydrocannabinol (THC), which is the active ingredient of marijuana. Suppression of CB1 is frequently suggested as a drug target or gene therapy for many conditions (e.g., obesity, Parkinson's disease). However, brain networks affected by CB1 remain elusive, and unanticipated psychological effects in a clinical trial had dire consequences. To better understand the whole brain effects of CB1 suppression we performed in vivo imaging on mice under complete knockout of the gene for CB1 (cnr1-/-) and also under the CB1 inverse agonist rimonabant. We examined white matter structural changes and brain function (network activity and directional uniformity) in cnr1-/- mice. In cnr1-/- mice, white matter (in both sexes) and functional directional uniformity (in male mice) were altered across the brain but network activity was largely unaltered. Conversely, under rimonabant, functional directional uniformity was not altered but network activity was altered in cortical regions, primarily in networks known to be altered by THC (e.g., neocortex, hippocampal formation). However, rimonabant did not alter many brain regions found in both our cnr1-/- results and previous behavioral studies of cnr1-/- mice (e.g., thalamus, infralimbic area). This suggests that chronic loss of cnr1 is substantially different from short-term suppression, subtly rewiring the brain but largely maintaining the network activity. Our results help explain why pathological mutations in CB1 (e.g., chronic pain) do not always provide insight into the side effects of CB1 suppression (e.g., clinical depression), and thus urge more preclinical studies for any drugs that suppress CB1.

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Energy metabolism is fundamental for life. It encompasses the utilization of carbohydrates, lipids, and proteins for internal processes, while aberrant energy metabolism is implicated in many diseases. In the present study, using three-dimensional (3D) printing from polycarbonate via fused deposition modeling, we propose a multi-nuclear radiofrequency (RF) coil design with integrated 1H birdcage and interchangeable X-nuclei (2H, 13C, 23Na, and 31P) single-loop coils for magnetic resonance imaging (MRI)/magnetic resonance spectroscopy (MRS). The single-loop coil for each nucleus attaches to an arc bracket that slides unrestrictedly along the birdcage coil inner surface, enabling convenient switching among various nuclei and animal handling. Compared to a commercial 1H birdcage coil, the proposed 1H birdcage coil exhibited superior signal-excitation homogeneity and imaging signal-to-noise ratio (SNR). For X-nuclei study, prominent peaks in spectroscopy for phantom solutions showed excellent SNR, and the static and dynamic peaks of in vivo spectroscopy validated the efficacy of the coil design in structural imaging and energy metabolism detection simultaneously.

Differential Effect of Global Signal Regression Between Awake and Anesthetized Conditions in Mice.

Introduction: In resting-state functional magnetic resonance imaging (rs-fMRI) studies, global signal regression (GSR) is a controversial preprocessing strategy. It effectively eliminates global noise driven by motion and respiration but also can introduce artifacts and remove functionally relevant metabolic information. Most preclinical rs-fMRI studies are performed in anesthetized animals, and anesthesia will alter both metabolic and neuronal activity. Methods: In this study, we explored the effect of GSR on rs-fMRI data collected under anesthetized and awake state in mice (n = 12). We measured global signal amplitude, and also functional connectivity (FC), functional connectivity density (FCD) maps, and brain modularity, all commonly used data-driven analysis methods to quantify connectivity patterns. Results: We found that global signal amplitude was similar between the awake and anesthetized states. However, GSR had a different impact on connectivity networks and brain modularity changes between states. We demonstrated that GSR had a more prominent impact on the anesthetized state, with a greater decrease in functional connectivity and increased brain modularity. We classified mice using the change in amplitude of brain modularity coefficient (ΔQ) before and after GSR processing. The results revealed that, when compared with the largest ΔQ group, the smallest ΔQ group had increased FCD in the cortex region in both the awake and anesthetized states. This suggests differences in individual mice may affect how GSR differentially affects awake versus anesthetized functional connectivity. Discussion: This study suggests that, for rs-fMRI studies which compare different physiological states, researchers should use GSR processing with caution.

Ketamine Effects on Energy Metabolism, Functional Connectivity and Working Memory in Healthy Humans.

Working memory (WM) is a crucial resource for temporary memory storage and the guiding of ongoing behavior. N-methyl-D-aspartate glutamate receptors (NMDARs) are thought to support the neural underpinnings of WM. Ketamine is an NMDAR antagonist that has cognitive and behavioral effects at subanesthetic doses. To shed light on subanesthetic ketamine effects on brain function, we employed a multimodal imaging design, combining gas-free calibrated functional magnetic resonance imaging (fMRI) measurement of oxidative metabolism (CMRO 2 ), resting-state cortical functional connectivity assessed with fMRI, and WM-related fMRI. Healthy subjects participated in two scan sessions in a randomized, double-blind, placebo-controlled design. Ketamine increased CMRO 2 and cerebral blood flow (CBF) in prefrontal cortex (PFC) and other cortical regions. However, resting-state cortical functional connectivity was not affected. Ketamine did not alter CBF-CMRO 2 coupling brain-wide. Higher levels of basal CMRO 2 were associated with lower task-related PFC activation and WM accuracy impairment under both saline and ketamine conditions. These observations suggest that CMRO 2 and resting-state functional connectivity index distinct dimensions of neural activity. Ketamine’s impairment of WM-related neural activity and performance appears to be related to its ability to produce cortical metabolic activation. This work illustrates the utility of direct measurement of CMRO 2 via calibrated fMRI in studies of drugs that potentially affect neurovascular and neurometabolic coupling.

Sleep fMRI with simultaneous electrophysiology at 9.4 T in male mice.

Sleep is ubiquitous and essential, but its mechanisms remain unclear. Studies in animals and humans have provided insights of sleep at vastly different spatiotemporal scales. However, challenges remain to integrate local and global information of sleep. Therefore, we developed sleep fMRI based on simultaneous electrophysiology at 9.4 T in male mice. Optimized un-anesthetized mouse fMRI setup allowed manifestation of NREM and REM sleep, and a large sleep fMRI dataset was collected and openly accessible. State dependent global patterns were revealed, and state transitions were found to be global, asymmetrical and sequential, which can be predicted up to 17.8 s using LSTM models. Importantly, sleep fMRI with hippocampal recording revealed potentiated sharp-wave ripple triggered global patterns during NREM than awake state, potentially attributable to co-occurrence of spindle events. To conclude, we established mouse sleep fMRI with simultaneous electrophysiology, and demonstrated its capability by revealing global dynamics of state transitions and neural events.

Affinity selection of double-click triazole libraries for rapid discovery of allosteric modulators for GLP-1 receptor.

The recently developed double-click reaction sequence [G. Meng et al., Nature 574, 86-89 (2019)] is expected to vastly expand the number and diversity of synthetically accessible 1,2,3-triazole derivatives. However, it remains elusive how to rapidly navigate the extensive chemical space created by double-click chemistry for bioactive compound discovery. In this study, we selected a particularly challenging drug target, the glucagon-like-peptide-1 receptor (GLP-1R), to benchmark our new platform for the design, synthesis, and screening of double-click triazole libraries. First, we achieved a streamlined synthesis of customized triazole libraries on an unprecedented scale (composed of 38,400 new compounds). By interfacing affinity-selection mass spectrometry and functional assays, we identified a series of positive allosteric modulators (PAMs) with unreported scaffolds that can selectively and robustly enhance the signaling activity of the endogenous GLP-1(9-36) peptide. Intriguingly, we further revealed an unexpected binding mode of new PAMs which likely act as a molecular glue between the receptor and the peptide agonist. We anticipate the merger of double-click library synthesis with the hybrid screening platform allows for efficient and economic discovery of drug candidates or chemical probes for various therapeutic targets.

Concerted roles of LRRTM1 and SynCAM 1 in organizing prefrontal cortex synapses and cognitive functions.

Multiple trans-synaptic complexes organize synapse development, yet their roles in the mature brain and cooperation remain unclear. We analyzed the postsynaptic adhesion protein LRRTM1 in the prefrontal cortex (PFC), a region relevant to cognition and disorders. LRRTM1 knockout (KO) mice had fewer synapses, and we asked whether other synapse organizers counteract further loss. This determined that the immunoglobulin family member SynCAM 1 controls synapse number in PFC and was upregulated upon LRRTM1 loss. Combined LRRTM1 and SynCAM 1 deletion substantially lowered dendritic spine number in PFC, but not hippocampus, more than the sum of single KO impairments. Their cooperation extended presynaptically, and puncta of Neurexins, LRRTM1 partners, were less abundant in double KO (DKO) PFC. Electrophysiology and fMRI demonstrated aberrant neuronal activity in DKO mice. Further, DKO mice were impaired in social interactions and cognitive tasks. Our results reveal concerted roles of LRRTM1 and SynCAM 1 across synaptic, network, and behavioral domains.

A new deuterium-labeled compound [2,3,4,6,6'-2 H5 ]-D-glucose for deuterium magnetic resonance metabolic imaging.

Deuterium (2 H) magnetic resonance imaging is an emerging approach for noninvasively studying glucose metabolism in vivo, which is important for understanding pathogenesis and monitoring the progression of many diseases such as tumors, diabetes, and neurodegenerative diseases. However, the synthesis of 2 H-labeled glucose is costly because of the expensive raw substrates and the requirement for extreme reaction conditions, making the 2 H-labeled glucose rather expensive and unaffordable for clinic use. In this study, we present a new deuterated compound, [2,3,4,6,6'-2 H5 ]-D-glucose, with an approximate 10-fold reduction in production costs. The synthesis route uses cheaper raw substrate methyl-α-D-glucopyranoside, relies on mild reaction conditions (80°C), and has higher deuterium labeling efficiency. Magnetic resonance spectroscopy (MRS) and mass spectroscopy experiments confirmed the successful deuterium labeling in the compound. Animal studies demonstrated that the substrate could describe the glycolytic metabolism in a glioma rat model by quantifying the downstream metabolites through 2 H-MRS on an ultrahigh field system. Comparison of the glucose metabolism characteristics was carried out between [2,3,4,6,6'-2 H5 ]-D-glucose and commercial [6,6'-2 H2 ]-D-glucose in the animal studies. This cost-effective compound will help facilitate the clinical translation of deuterium magnetic resonance imaging, and enable this powerful metabolic imaging modality to be widely used in both preclinical and clinical research and applications.

An intensity-based post-processing tool for 3D instance segmentation of organelles in soft X-ray tomograms.

Investigating the 3D structures and rearrangements of organelles within a single cell is critical for better characterizing cellular function. Imaging approaches such as soft X-ray tomography have been widely applied to reveal a complex subcellular organization involving multiple inter-organelle interactions. However, 3D segmentation of organelle instances has been challenging despite its importance in organelle characterization. Here we propose an intensity-based post-processing tool to identify and separate organelle instances. Our tool separates sphere-like (insulin vesicle) and columnar-shaped organelle instances (mitochondrion) based on the intensity of raw tomograms, semantic segmentation masks, and organelle morphology. We validate our tool using synthetic tomograms of organelles and experimental tomograms of pancreatic ß-cells to separate insulin vesicle and mitochondria instances. As compared to the commonly used connected regions labeling, watershed, and watershed + Gaussian filter methods, our tool results in improved accuracy in identifying organelles in the synthetic tomograms and an improved description of organelle structures in ß-cell tomograms. In addition, under different experimental treatment conditions, significant changes in volumes and intensities of both insulin vesicle and mitochondrion are observed in our instance results, revealing their potential roles in maintaining normal ß-cell function. Our tool is expected to be applicable for improving the instance segmentation of other images obtained from different cell types using multiple imaging modalities.

Thalamic activations in rat brain by fMRI during tactile (forepaw, whisker) and non-tactile (visual, olfactory) sensory stimulations.

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

Aerobic glycolysis imaging of epileptic foci during the inter-ictal period.

BACKGROUND: In drug-resistant epilepsy, surgical resection of the epileptic focus can end seizures. However, success is dependent on the ability to identify foci locations and, unfortunately, current methods like electrophysiology and positron emission tomography can give contradictory results. During seizures, glucose is metabolized at epileptic foci through aerobic glycolysis, which can be imaged through the oxygen-glucose index (OGI) biomarker. However, inter-ictal (between seizures) OGI changes have not been studied, which has limited its application. METHODS: 18 healthy controls and 24 inter-ictal, temporal lobe epilepsy patients underwent simultaneous positron emission tomography (PET) and magnetic resonance imaging (MRI) scans. We used [18F]fluorodeoxyglucose-PET (FDG-PET) to detect cerebral glucose metabolism, and calibrated functional MRI to acquire relative oxygen consumption. With these data, we calculated relative OGI maps. FINDINGS: While bilaterally symmetrical in healthy controls, we observed, in patients during the inter-ictal period, higher OGI ipsilateral to the epileptic focus than contralateral. While traditional FDG-PET results and temporal lobe OGI results usually both agreed with invasive electrophysiology, in cases where FDG-PET disagreed with electrophysiology, temporal lobe OGI agreed with electrophysiology, and vice-versa. INTERPRETATION: As either our novel epilepsy biomarker or traditional approaches located foci in every case, our work provides promising insights into metabolic changes in epilepsy. Our method allows single-session OGI measurement which can be useful in other diseases. FUNDING: This work was supported by ShanghaiTech University, the Shanghai Municipal Government, the National Natural Science Foundation of China Grant (No. 81950410637) and Shanghai Municipal Key Clinical Specialty (No. shslczdzk03403). F. H. and P. H. were supported by USA National Institute of Health grants (R01 NS-100106, R01 MH-067528).Z. W. was supported by the Key-Area Research and Development Program of Guangdong Province (2019B030335001), National Natural Science Foundation of China (No. 82151303), and National Key R&D Program of China (No. 2021ZD0204002).

Proton/Deuterium Magnetic Resonance Imaging of Rodents at 9.4T Using Birdcage Coils.

The purpose of the present study was to fabricate a volume coil for proton/deuterium magnetic resonance imaging (MRI) in rodents at 9.4 T. Two birdcage radiofrequency (RF) coils have been designed for proton/deuterium MRI: the rungs of two concentric birdcages were azimuthally interleaved with each other for better decoupling, and the two coils were tuned to 400.3 and 61.4 MHz for 1 H/2 H resonance at 9.4 T. Compared to a commercially available coil, the proposed 1 H/2 H RF coil provides reasonable transmission efficiency and imaging signal-to-noise ratio (SNR); the relationships among imaging parameters such as SNR, voxel size, and deuterium oxide concentrations have been quantitatively studied, and the linear correlation results together with the spectroscopic data in vivo indicate its feasibility in deuterium metabolic imaging (DMI) in vivo. Our study indicates that using the birdcage design for MRI signal excitation combined with surface coil array for signal reception can facilitate DMI investigations more effectively towards future pre-clinical and clinical applications. As a noninvasive method by measuring nonhydrogen nuclear deuterium signals to reflect metabolite information, DMI will feature prominently in future precision medicine through the whole process of diagnosis, treatment, and prognosis. © 2021 Bioelectromagnetics Society.

High-resolution relaxometry-based calibrated fMRI in murine brain: Metabolic differences between awake and anesthetized states.

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

Longitudinal neural connection detection using a ferritin-encoding adeno-associated virus vector and in vivo MRI method.

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

Organized cannabinoid receptor distribution in neurons revealed by super-resolution fluorescence imaging.

G-protein-coupled receptors (GPCRs) play important roles in cellular functions. However, their intracellular organization is largely unknown. Through investigation of the cannabinoid receptor 1 (CB1), we discovered periodically repeating clusters of CB1 hotspots within the axons of neurons. We observed these CB1 hotspots interact with the membrane-associated periodic skeleton (MPS) forming a complex crucial in the regulation of CB1 signaling. Furthermore, we found that CB1 hotspot periodicity increased upon CB1 agonist application, and these activated CB1 displayed less dynamic movement compared to non-activated CB1. Our results suggest that CB1 forms periodic hotspots organized by the MPS as a mechanism to increase signaling efficacy upon activation.

Orthonasal versus retronasal glomerular activity in rat olfactory bulb by fMRI.

Odorants can reach olfactory receptor neurons (ORNs) by two routes: orthonasally, when volatiles enter the nasal cavity during inhalation/sniffing, and retronasally, when food volatiles released in the mouth pass into the nasal cavity during exhalation/eating. Previous work in humans has shown that both delivery routes of the same odorant can evoke distinct perceptions and patterns of neural responses in the brain. Each delivery route is known to influence specific responses across the dorsal region of the glomerular sheet in the olfactory bulb (OB), but spatial distributions across the entire glomerular sheet throughout the whole OB remain largely unexplored. We used functional MRI (fMRI) to measure and compare activations across the entire glomerular sheet in rat OB resulting from both orthonasal and retronasal stimulations of the same odors. We observed reproducible fMRI activation maps of the whole OB during both orthonasal and retronasal stimuli. However, retronasal stimuli required double the orthonasal odor concentration for similar response amplitudes. Regardless, both the magnitude and spatial extent of activity were larger during orthonasal versus retronasal stimuli for the same odor. Orthonasal and retronasal response patterns show overlap as well as some route-specific dominance. Orthonasal maps were dominant in dorsal-medial regions, whereas retronasal maps were dominant in caudal and lateral regions. These different whole OB encodings likely underlie differences in odor perception between these biologically important routes for odorants among mammals. These results establish the relationships between orthonasal and retronasal odor representations in the rat OB.

Disentangling Multispectral Functional Connectivity With Wavelets.

The field of brain connectomics develops our understanding of the brain's intrinsic organization by characterizing trends in spontaneous brain activity. Linear correlations in spontaneous blood-oxygen level dependent functional magnetic resonance imaging (BOLD-fMRI) fluctuations are often used as measures of functional connectivity (FC), that is, as a quantity describing how similarly two brain regions behave over time. Given the natural spectral scaling of BOLD-fMRI signals, it may be useful to represent BOLD-fMRI as multiple processes occurring over multiple scales. The wavelet domain presents a transform space well suited to the examination of multiscale systems as the wavelet basis set is constructed from a self-similar rescaling of a time and frequency delimited kernel. In the present study, we utilize wavelet transforms to examine fluctuations in whole-brain BOLD-fMRI connectivity as a function of wavelet spectral scale in a sample (N = 31) of resting healthy human volunteers. Information theoretic criteria measure relatedness between spectrally-delimited FC graphs. Voxelwise comparisons of between-spectra graph structures illustrate the development of preferential functional networks across spectral bands.

Impact of Global Mean Normalization on Regional Glucose Metabolism in the Human Brain.

Because the human brain consumes a disproportionate fraction of the resting body's energy, positron emission tomography (PET) measurements of absolute glucose metabolism (CMRglc) can serve as disease biomarkers. Global mean normalization (GMN) of PET data reveals disease-based differences from healthy individuals as fractional changes across regions relative to a global mean. To assess the impact of GMN applied to metabolic data, we compared CMRglc with and without GMN in healthy awake volunteers with eyes closed (i.e., control) against specific physiological/clinical states, including healthy/awake with eyes open, healthy/awake but congenitally blind, healthy/sedated with anesthetics, and patients with disorders of consciousness. Without GMN, global CMRglc alterations compared to control were detected in all conditions except in congenitally blind where regional CMRglc variations were detected in the visual cortex. However, GMN introduced regional and bidirectional CMRglc changes at smaller fractions of the quantitative delocalized changes. While global information was lost with GMN, the quantitative approach (i.e., a validated method for quantitative baseline metabolic activity without GMN) not only preserved global CMRglc alterations induced by opening eyes, sedation, and varying consciousness but also detected regional CMRglc variations in the congenitally blind. These results caution the use of GMN upon PET-measured CMRglc data in health and disease.

Amygdala hyper-connectivity in a mouse model of unpredictable early life stress.

Childhood maltreatment is associated with a wide range of psychopathologies including anxiety that emerge in childhood and in many cases persist in adulthood. Increased amygdala activation in response to threat and abnormal amygdala connectivity with frontolimbic brain regions, such as the hippocampus and the prefrontal cortex, are some of the most consistent findings seen in individuals exposed to childhood maltreatment. The underlying mechanisms responsible for these changes are difficult to study in humans but can be elucidated using animal models of early-life stress. Such studies are especially powerful in the mouse where precise control of the genetic background and the stress paradigm can be coupled with resting-state fMRI (rsfMRI) to map abnormal connectivity in circuits that regulate anxiety. To address this issue we first compared the effects of two models of early-life stress, limited bedding (LB) and unpredictable postnatal stress (UPS), on anxiety-like behavior in juvenile and adult mice. We found that UPS, but not LB, causes a robust increase in anxiety in juvenile and adult male mice. Next, we used rsfMRI to compare frontolimbic connectivity in control and UPS adult male mice. We found increased amygdala-prefrontal cortex and amygdala-hippocampus connectivity in UPS. The strength of the amygdala-hippocampal and amygdala-prefrontal cortex connectivity was highly correlated with anxiety-like behavior in the open-field test and elevated plus maze. These findings are the first to link hyperconnectivity in frontolimbic circuits and increased anxiety in a mouse model of early-life stress, allowing for more mechanistic understanding of parallel findings in humans.