Functional ultrasound imaging (fUSI) is an emerging hemodynamic neuroimaging modality whose potential for connectome-scale network mapping remains largely untested. Here, we establish a non-invasive transcranial fUSI protocol and an fMRI-inspired preprocessing framework that enable robust resting-state functional connectomics in the mouse. We show that fUSI resolves canonical brain-wide cortical and subcortical networks with high spatial concordance to fMRI, including a default-mode (DMN) and a laterocortical network. Notably, fUSI networks are robustly embedded within the structural connectome, with structure-function coupling being parsimoniously described by four dominant axes that differentially relate functional systems to known anatomical substrates. We also show that, beyond static organization, fUSI reproduces hallmark resting state fMRI dynamics, including dominant anti-correlated coactivation patterns (CAPs) and a structured transition architecture that converges onto three stable attractor modes. Together, these results establish transcranial fUSI as a portable and scalable complement to fMRI for connectome-scale mapping of mouse brain networks. ### Competing Interest Statement The authors have declared no competing interest. European Research Council (ERC), 802371, 101125054 Brain and Machines Flagship Programme of the Italian Institute of Technology

Driven by a period of accelerated progress and recent technical breakthroughs, whole-brain functional neuroimaging in rodents offers exciting new possibilities for addressing basic questions about brain function and its alterations. In response to lessons learned from the human neuroimaging community, leading scientists and researchers in the field convened to address existing barriers and outline ambitious goals for the future. This article captures these discussions, highlighting a shared vision to advance rodent functional neuroimaging into an era of increased impact.

Chromosome 22q11.2 deletion increases the risk of neuropsychiatric disorders like autism and schizophrenia. Disruption of large-scale functional connectivity in 22q11 deletion syndrome (22q11DS) has been widely reported, but the biological factors driving these changes remain unclear. We used a cross-species design to uncover the developmental trajectory and neural underpinnings of brain dysconnectivity in 22q11DS. In LgDel mice, a model for 22q11DS, we found age-specific patterns of brain dysconnectivity, with widespread fMRI hyperconnectivity in juvenile mice reconfiguring to hippocampal hypoconnectivity over puberty. These changes correlated with developmental alterations in dendritic spine density, and both were transiently normalized by GSK3β inhibition, suggesting a synaptic origin for this phenomenon. Notably, analogous pubertal hyperconnectivity-to-hypoconnectivity reconfiguration occurs in human 22q11DS, affecting cortical regions enriched for GSK3β-associated synaptic genes and autism-relevant transcripts. This dysconnectivity also predicts age-dependent social alterations in 22q11DS individuals. These results suggest that synaptic mechanisms underlie developmental brain dysconnectivity in 22q11DS.

Chromosome 22q11.2 deletion is among the strongest known genetic risk factors for neuropsychiatric disorders, including autism and schizophrenia. Brain imaging studies have reported disrupted large-scale functional connectivity in people with 22q11 deletion syndrome (22q11DS). However, the significance and biological determinants of these functional alterations remain unclear. Here, we use a cross-species design to investigate the developmental trajectory and neural underpinnings of brain dysconnectivity in 22q11DS. We find that LgDel mice, an established mouse model of 22q11DS, exhibit age-specific patterns of functional MRI (fMRI) dysconnectivity, with widespread fMRI hyper-connectivity in juvenile mice reverting to focal hippocampal hypoconnectivity over puberty. These fMRI connectivity alterations are mirrored by co-occurring developmental alterations in dendritic spine density, and are both transiently normalized by developmental GSK3β inhibition, suggesting a synaptic origin for this phenomenon. Notably, analogous hyper-to hypoconnectivity reconfiguration occurs also in human 22q11DS, where it affects hippocampal and cortical regions spatially enriched for synaptic genes that interact with GSK3β, and autism-relevant transcripts. Functional dysconnectivity in somatomotor components of this network is predictive of age-dependent social alterations in 22q11.2 deletion carriers. Taken together, these findings suggest that synaptic-related mechanisms underlie developmentally mediated functional dysconnectivity in 22q11DS.

While shaped and constrained by axonal connections, fMRI-based functional connectivity reorganizes in response to varying interareal input or pathological perturbations. However, the causal contribution of regional brain activity to whole-brain fMRI network organization remains unclear. Here we combine neural manipulations, resting-state fMRI and in vivo electrophysiology to probe how inactivation of a cortical node causally affects brain-wide fMRI coupling in the mouse. We find that chronic inhibition of the medial prefrontal cortex (PFC) via overexpression of a potassium channel increases fMRI connectivity between the inhibited area and its direct thalamo-cortical targets. Acute chemogenetic inhibition of the PFC produces analogous patterns of fMRI overconnectivity. Using in vivo electrophysiology, we find that chemogenetic inhibition of the PFC enhances low frequency (0.1–4 Hz) oscillatory power via suppression of neural firing not phase-locked to slow rhythms, resulting in increased slow and δ band coherence between areas that exhibit fMRI overconnectivity. These results provide causal evidence that cortical inactivation can counterintuitively increase fMRI connectivity via enhanced, less-localized slow oscillatory processes.

To support frequency-coded information transfer, mammalian synapses tightly synchronize neurotransmitter release to action potentials (APs). However, release desynchronizes during AP trains, especially at room temperature. Here we show that suppression of asynchronous release by Synaptotagmin-1 (Syt1), but not release triggering, is highly temperature sensitive, and enhances synchronous release during high-frequency stimulation. In Syt1-deficient synapses, asynchronous release increased with temperature, opposite to wildtype synapses. Mutations in Syt1 C2B-domain polybasic stretch (Syt1 K326Q,K327Q,K331Q) did not affect synchronization during sustained activity, while the previously observed reduced synchronous response to a single AP was confirmed. However, an inflexible linker between the C2-domains (Syt1 9Pro) reduced suppression, without affecting synchronous release upon a single AP. Syt1 9Pro expressing synapses showed impaired synchronization during AP trains, which was rescued by buffering global Ca2+ to prevent asynchronous release. Hence, frequency coding relies on Syt1's temperature sensitive suppression of asynchronous release, an aspect distinct from its known vesicle recruitment and triggering functions.