An imaging-coupled 3D printing methodology for the design, optimization, and fabrication of a customized nerve repair technology for complex injuries is presented. The custom scaffolds are deterministically fabricated via a microextrusion printing principle which enables the simultaneous incorporation of anatomical geometries, biomimetic physical cues, and spatially controlled biochemical gradients in a one-pot 3D manufacturing approach.

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.

Highlights d Ambient warmth activates DRN Vgat neurons d DRN Vgat neurons regulate energy expenditure through locomotion and thermogenesis d DRN Vgat neurons exhibit vast projections and polysynaptically innervate brown fat d DRN Vgat projections differentially regulate food intake and energy expenditure

The use of viruses as transneuronal tracers has become an increasingly powerful technique for defining the synaptic organization of neural networks. Although a number of recombinant alpha herpesviruses are known to spread selectively in the retrograde direction through neural circuits only one strain, the H129 strain of herpes simplex virus type 1, is reported to selectively spread in the anterograde direction. However, it is unclear from the literature whether there is an absolute block or an attenuation of retrograde spread of H129. Here we demonstrate efficient anterograde spread, and temporally delayed retrograde spread, of H129 and three novel recombinants. In vitro studies revealed no differences in anterograde and retrograde spread of parental H129 and its recombinants through superior cervical ganglion neurons. In vivo injections of rat striatum revealed a clear bias of anterograde spread, although evidence of deficient retrograde transport was also present. Evidence of temporally delayed retrograde transneuronal spread of H129 in the retina was observed following injection of the lateral geniculate nucleus. The data also demonstrated that three novel recombinants efficiently express unique fluorescent reporters and have the capacity to infect the same neurons in dual infection paradigms. From these experiments we conclude that H129 and its recombinants efficiently infect neurons through anterograde transneuronal passage, but also are capable of temporally delayed retrograde transneuronal spread. In addition, the capacity to produce dual infection of projection targets following anterograde transneuronal passage provides an important addition to viral transneuronal tracing technology.

Highlights d Cellular resolution imaging of IL-NAc projection neurons in rats d IL-NAc neurons display spatial and temporal selectivity during drug seeking d After a drug-free period, drug motivation is high and neural coding is degraded

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In the nearly two decades since the popularization of green fluorescent protein (GFP), fluorescent protein-based methodologies have revolutionized molecular and cell biology, allowing us to literally see biological processes as never before. Naturally, this revolution has extended to virology in general, and to the study of alpha herpesviruses in particular. In this review, we provide a compendium of reported fluorescent protein fusions to herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV) structural proteins, discuss the underappreciated challenges of fluorescent protein-based approaches in the context of a replicating virus, and describe general strategies and best practices for creating new fluorescent fusions. We compare fluorescent protein methods to alternative approaches, and review two instructive examples of the caveats associated with fluorescent protein fusions, including describing several improved fluorescent capsid fusions in PRV. Finally, we present our future perspectives on the types of powerful experiments these tools now offer.

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...