SUMMARY Cerebellar outputs take polysynaptic routes to reach the rest of the brain, impeding conventional tracing. Here, we quantify pathways between the cerebellum and forebrain by using transsynaptic tracing viruses and a whole-brain analysis pipeline. With retrograde tracing, we find that most descending paths originate from the somatomotor cortex. Anterograde tracing of ascending paths encompasses most thalamic nuclei, especially ventral posteromedial, lateral posterior, mediodorsal, and reticular nuclei. In the neocortex, sensorimotor regions contain the most labeled neurons, but we find higher densities in associative areas, including orbital, anterior cingulate, prelimbic, and infralimbic cortex. Patterns of ascending expression correlate with c-Fos expression after optogenetic inhibition of Purkinje cells. Our results reveal homologous networks linking single areas of the cerebellar cortex to diverse forebrain targets. We conclude that shared areas of the cerebellum are positioned to provide sensory-motor information to regions implicated in both movement and nonmotor function.

16Cerebellar outputs take multisynaptic paths to reach higher brain areas, impeding tracing 17 efforts. Here we quantify pathways between cerebellum and contralateral 18 thalamic/corticostriatal structures using the anterograde transsynaptic tracer herpes 19 simplex virus type 1 (H129), the retrograde tracer pseudorabies virus (Bartha), adeno-20 associated virus, and a whole-brain pipeline for neuron-level analysis using light-sheet 21 microscopy. In ascending pathways, sensorimotor regions contained the most labeled 22 neurons, but higher densities were found in associative areas, including orbital, anterior 23 cingulate, prelimbic, and infralimbic cortex. Ascending paths passed through most 24 thalamic nuclei, especially ventral posteromedial and lateral posterior (sensorimotor), 25 mediodorsal (associative), and reticular (modulatory) nuclei. Retrograde tracing revealed 26 descending paths originating largely from somatomotor cortex. Patterns of ascending 27 influence correlated with anatomical pathway strengths, as measured by brainwide 28 mapping of c-Fos responses to optogenetic inhibition of Purkinje cells. Our results 29 reveal parallel functional networks linking cerebellum to forebrain and suggest that 30 cerebellum uses sensory-motor information to guide both movement and nonmotor 31 functions. 32 most mammalian brains 8 . The major descending corticocerebellar pathway passes through the 41 pons and the majority of returning ascending fibers pass through the thalamus 9,10 , comprising 42 two massive within-brain long-distance pathways 11 . Other polysynaptic pathways exist between 43 the cerebellum and neocortex, including a smaller ascending pathway through ventral tegmental 44 area that has attracted recent interest 12 . These descending and ascending pathways are 45 suggested to form closed loops 13 , giving each cerebellar region one or more specific neocortical 46 partners with which it exchanges information. 47This picture lacks critical information: the identity of those distant regions, which have 48 been difficult to map. Given the brain-wide nature of cerebello-cortical pathways, researchers 49 have used large-scale approaches to examine the functional significance of these pathways. 50Transcranial magnetic stimulation in humans demonstrated that the cerebellum influences 51 neocortical excitability 14 , including cognitive and affective circuits 15 . Functional MRI can attain 52 subcentimeter resolution, detect long-distance correlations 16 , and when coupled with cerebellar 53 stimulation, demonstrate causal relationships 17 . Functional imaging at cellular resolution in 54 nonhuman animals has been made possible by visualizing c-Fos, an immediate-early gene 55 product whose expression is regulated by neural activity. Although useful in demonstrating 56 communication with distant brain regions, these methods do not provide cellular-resolution 57 information about cerebello-cortical circuits. 58Pathways entering and exiting the cerebellum pass through synapses in the brainstem 59 and the cer...

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Recombinant adeno-associated viruses (rAAVs) are used as gene therapy vectors to treat central nervous system (CNS) diseases. Despite their safety and broad tropism, important issues need to be corrected such as the limited payload capacity and the lack of small gene promoters providing long-term, panneuronal transgene expression in the CNS. Commonly used gene promoters are relatively large and can be repressed a few months after CNS transduction, risking the long-term performance of single-dose gene therapy applications. We used a whole-CNS screening approach based on systemic delivery of AAV-PHP.eB, iDisco+ tissue-clearing and light-sheet microscopy to identify three small latency-associated promoters (LAPs) from the herpesvirus pseudorabies virus (PRV). These promoters are LAP1 (404 bp), LAP2 (498 bp), and LAP1_2 (880 bp). They drive chronic transcription of the virus-encoded latency-associated transcript (LAT) during productive and latent phases of PRV infection. We observed stable, panneuronal transgene transcription and translation from AAV-LAPs in the CNS for 6 months post AAV transduction. In several CNS areas, the number of cells expressing the transgene was higher for LAP2 than the large conventional EF1a promoter (1,264 bp). Our data suggest that the LAPs are suitable candidates for viral vector-based CNS gene therapies requiring chronic transgene expression after one-time viral-vector administration.

The vascular endothelium plays a critical role in the health and disease of the cardiovascular system. Importantly, biomechanical stimuli generated by blood flow and sensed by the endothelium constitute important local inputs that are translated into transcriptional programs and functional endothelial phenotypes. Pulsatile, laminar flow, characteristic of regions in the vasculature that are resistant to atherosclerosis, evokes an atheroprotective endothelial phenotype. This atheroprotective phenotype is integrated by the transcription factor Kruppel-like factor-2 (KLF2), and therefore the expression of KLF2 can be used as a proxy for endothelial atheroprotection. Here, we report the generation and characterization of a cellular KLF2 reporter system, based on green fluorescence protein (GFP) expression driven by the human KLF2 promoter. This reporter is induced selectively by an atheroprotective shear stress waveform in human endothelial cells, is regulated by endogenous signaling events, and is activated by the pharmacological inducer of KLF2, simvastatin, in a dose-dependent manner. This reporter system can now be used to probe KLF2 signaling and for the discovery of a novel chemical-biological space capable of acting as the "pharmacomimetics of atheroprotective flow" on the vascular endothelium.

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The cerebellum regulates nonmotor behavior, but the routes of influence are not well characterized. Here we report a necessary role for the posterior cerebellum in guiding a reversal learning task through a network of diencephalic and neocortical structures, and in flexibility of free behavior. After chemogenetic inhibition of lobule VI vermis or hemispheric crus I Purkinje cells, mice could learn a water Y-maze but were impaired in ability to reverse their initial choice. To map targets of perturbation, we imaged c-Fos activation in cleared whole brains using light-sheet microscopy. Reversal learning activated diencephalic and associative neocortical regions. Distinctive subsets of structures were altered by perturbation of lobule VI (including thalamus and habenula) and crus I (including hypothalamus and prelimbic/orbital cortex), and both perturbations influenced anterior cingulate and infralimbic cortex. To identify functional networks, we used correlated variation in c-Fos activation within each group. Lobule VI inactivation weakened within-thalamus correlations, while crus I inactivation divided neocortical activity into sensorimotor and associative subnetworks. In both groups, high-throughput automated analysis of whole-body movement revealed deficiencies in across-day behavioral habituation to an open-field environment. Taken together, these experiments reveal brainwide systems for cerebellar influence that affect multiple flexible responses.

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