Optimization of the Fluorescent Protein Expression Level Based on Pseudorabies Virus Bartha Strain for Neural Circuit Tracing.
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ABSTRACT:
SUBMITTER: Jia F
PROVIDER: S-EPMC6597954 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The study of coordinated activity in neuronal circuits has been challenging without a method to simultaneously report activity and connectivity. Here we present the first use of pseudorabies virus (PRV), which spreads through synaptically connected neurons, to express a fluorescent calcium indicator protein and monitor neuronal activity in a living animal. Fluorescence signals were proportional to action potential number and could reliably detect single action potentials in vitro. With two-photon imaging in vivo, we observed both spontaneous and stimulated activity in neurons of infected murine peripheral autonomic submandibular ganglia (SMG). We optically recorded the SMG response in the salivary circuit to direct electrical stimulation of the presynaptic axons and to physiologically relevant sensory stimulation of the oral cavity. During a time window of 48 hours after inoculation, few spontaneous transients occurred. By 72 hours, we identified more frequent and prolonged spontaneous calcium transients, suggestive of neuronal or tissue responses to infection that influence calcium signaling. Our work establishes in vivo investigation of physiological neuronal circuit activity and subsequent effects of infection with single cell resolution.
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Project description:The widely used pseudorabies virus (PRV) Bartha-K61 vaccine has played a key role in the eradication of PRV. Since late 2011, however, a disease characterized by neurologic symptoms and a high number of deaths among newborn piglets has occurred among Bartha-K61-vaccinated pigs on many farms in China. Clinical samples from pigs on 15 farms in 6 provinces were examined. The PRV gE gene was detectable by PCR in all samples, and sequence analysis of the gE gene showed that all isolates belonged to a relatively independent cluster and contained 2 amino acid insertions. A PRV (named HeN1) was isolated and caused transitional fever in pigs. In protection assays, Bartha-K61 vaccine provided 100% protection against lethal challenge with SC (a classical PRV) but only 50% protection against 4 challenges with strain HeN1. The findings suggest that Bartha-K61 vaccine does not provide effective protection against PRV HeN1 infection.
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Project description:Monosynaptic rabies virus tracing is a unique and powerful tool used to identify neurons making direct presynaptic connections onto neurons of interest across the entire nervous system. Current methods utilize complementation of glycoprotein gene-deleted rabies of the SAD B19 strain with its glycoprotein, B19G, to mediate retrograde transsynaptic spread across a single synaptic step. In most conditions, this method labels only a fraction of input neurons and would thus benefit from improved efficiency of transsynaptic spread. Here, we report newly engineered glycoprotein variants to improve transsynaptic efficiency. Among them, oG (optimized glycoprotein) is a codon-optimized version of a chimeric glycoprotein consisting of the transmembrane/cytoplasmic domain of B19G and the extracellular domain of rabies Pasteur virus strain glycoprotein. We demonstrate that oG increases the tracing efficiency for long-distance input neurons up to 20-fold compared to B19G. oG-mediated rabies tracing will therefore allow identification and study of more complete monosynaptic input neural networks.
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Project description:Pseudorabies virus (PRV) is a member of Alphaherpesvirinae subfamily, its neurotropism and latency infection attract the attention of many scientists. PRV tagged with a fluorescent reporter gene as a tracker has been used to analyze neuronal circuits, including anterograde and retrograde. In this study, we used fosmid library to construct a rapid and efficient platform to generate recombinant PRV. Firstly, the highly purified PRV ShuangCheng (SC) genomic DNA was sheared randomly into approximately 30-49-kb DNA fragments. After end-blunting and phosphorylation, the DNA fragments were cloned into the fosmid vector and transformed into Escherichia coli. A total of 200 fosmids that cover the complete genome of PRV SC was sequenced. Thirteen fosmid combinations in five groups were transfected into Vero cells, respectively, and each group can successfully rescue PRV strain SC. There was no significant difference between wild type and recombinant in both morphology and growth kinetics. In the next step, an enhanced green fluorescent protein (EGFP) was fused into the amino-terminal of UL36 protein by Red/ET recombination technology, and recombinant rPRV SC-UL36-EGFP was rescued successfully. At last, the single viral particles with green fluorescent were monitored retrograde moving in the axon with an average velocity of 0.71 ± 0.43 μm/s at 0.5-2 h post infection (hpi) and anterograde moving with an average velocity of 0.75 ± 0.49 μm/s at eight hpi. Integration of fosmid library and Red/ET recombination technology in our work was highly efficient and stable for constructing PRV recombinants. This study will accelerate understanding the biology of PRV and the development of novel vaccines.
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Project description:The mammalian nervous system is comprised of a seemingly infinitely complex network of specialized synaptic connections that coordinate the flow of information through it. The field of connectomics seeks to map the structure that underlies brain function at resolutions that range from the ultrastructural, which examines the organization of individual synapses that impinge upon a neuron, to the macroscopic, which examines gross connectivity between large brain regions. At the mesoscopic level, distant and local connections between neuronal populations are identified, providing insights into circuit-level architecture. Although neural tract tracing techniques have been available to experimental neuroscientists for many decades, considerable methodological advances have been made in the last 20 years due to synergies between the fields of molecular biology, virology, microscopy, computer science and genetics. As a consequence, investigators now enjoy an unprecedented toolbox of reagents that can be directed against selected subpopulations of neurons to identify their efferent and afferent connectomes. Unfortunately, the intersectional nature of this progress presents newcomers to the field with a daunting array of technologies that have emerged from disciplines they may not be familiar with. This review outlines the current state of mesoscale connectomic approaches, from data collection to analysis, written for the novice to this field. A brief history of neuroanatomy is followed by an assessment of the techniques used by contemporary neuroscientists to resolve mesoscale organization, such as conventional and viral tracers, and methods of selecting for sub-populations of neurons. We consider some weaknesses and bottlenecks of the most widely used approaches for the analysis and dissemination of tracing data and explore the trajectories that rapidly developing neuroanatomy technologies are likely to take.
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Project description:[This corrects the article DOI: 10.3389/fnins.2019.00897.].
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Project description:Neural circuits interconnect to organize large-scale networks that generate perception, cognition, memory, and behavior. Information in the nervous system is processed both through parallel, independent circuits and through intermixing circuits. Analyzing the interaction between circuits is particularly indispensable for elucidating how the brain functions. Monosynaptic circuit tracing with glycoprotein (G) gene-deleted rabies viral vectors (RVΔG) comprises a powerful approach for studying the structure and function of neural circuits. Pseudotyping of RVΔG with the foreign envelope EnvA permits expression of transgenes such as fluorescent proteins, genetically-encoded sensors, or optogenetic tools in cells expressing TVA, a cognate receptor for EnvA. Trans-complementation with rabies virus glycoproteins (RV-G) enables trans-synaptic labeling of input neurons directly connected to the starter neurons expressing both TVA and RV-G. However, it remains challenging to simultaneously map neuronal connections from multiple cell populations and their interactions between intermixing circuits solely with the EnvA/TVA-mediated RV tracing system in a single animal. To overcome this limitation, here, we multiplexed RVΔG circuit tracing by optimizing distinct viral envelopes (oEnvX) and their corresponding receptors (oTVX). Based on the EnvB/TVB and EnvE/DR46-TVB systems derived from the avian sarcoma leukosis virus (ASLV), we developed optimized TVB receptors with lower or higher affinity (oTVB-L or oTVB-H) and the chimeric envelope oEnvB, as well as an optimized TVE receptor with higher affinity (oTVE-H) and its chimeric envelope oEnvE. We demonstrated independence of RVΔG infection between the oEnvA/oTVA, oEnvB/oTVB, and oEnvE/oTVE systems and in vivo proof-of-concept for multiplex circuit tracing from two distinct classes of layer 5 neurons targeting either other cortical or subcortical areas. We also successfully labeled common input of the lateral geniculate nucleus to both cortico-cortical layer 5 neurons and inhibitory neurons of the mouse V1 with multiplex RVΔG tracing. These oEnvA/oTVA, oEnvB/oTVB, and oEnvE/oTVE systems allow for differential labeling of distinct circuits to uncover the mechanisms underlying parallel processing through independent circuits and integrated processing through interaction between circuits in the brain.
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Project description:Alzheimer's disease (AD) is a progressive neurodegenerative disorder with growing major health impacts, particularly in countries with aging populations. The examination of neural circuit mechanisms in AD mouse models is a recent focus for identifying new AD treatment strategies. We hypothesize that age-progressive changes of both long-range and local hippocampal neural circuit connectivity occur in AD. Recent advancements in viral-genetic technologies provide new opportunities for semi-quantitative mapping of cell-type-specific neural circuit connections in AD mouse models. We applied a recently developed monosynaptic rabies tracing method to hippocampal neural circuit mapping studies in AD model mice to determine how local and global circuit connectivity to hippocampal CA1 excitatory neurons may be altered in the single APP knock-in (APP-KI) AD mouse model. To determine age-related AD progression, we measured circuit connectivity in age-matched littermate control and AD model mice at two different ages (3-4 vs. 10-11 months old). We quantitatively mapped the connectivity strengths of neural circuit inputs to hippocampal CA1 excitatory neurons from brain regions including hippocampal subregions, medial septum, subiculum and entorhinal cortex, comparing different age groups and genotypes. We focused on hippocampal CA1 because of its clear relationship with learning and memory and that the hippocampal formation shows clear neuropathological changes in human AD. Our results reveal alterations in circuit connectivity of hippocampal CA1 in AD model mice. Overall, we find weaker extrinsic CA1 input connectivity strengths in AD model mice compared with control mice, including sex differences of reduced subiculum to CA1 inputs in aged female AD mice compared with aged male AD mice. Unexpectedly, we find a connectivity pattern shift with an increased proportion of inputs from the CA3 region to CA1 excitatory neurons when comparing young and old AD model mice, as well as old wild-type mice and old AD model mice. These unexpected shifts in CA3-CA1 input proportions in this AD mouse model suggest the possibility that compensatory circuit increases may occur in response to connectivity losses in other parts of the hippocampal circuits. We expect that this work provides new insights into the neural circuit mechanisms of AD pathogenesis.
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Project description:Brain functions are accomplished by polysynaptic circuits formed by neurons wired together through multiple orders of synaptic connections. Polysynaptic connectivity has been difficult to examine due to a lack of methods of continuously tracing the pathways in a controlled manner. Here, we demonstrate directed, stepwise retrograde polysynaptic tracing by inducible reconstitution of replication-deficient trans-neuronal pseudorabies virus (PRVΔIE) in the brain. Furthermore, PRVΔIE replication can be temporally restricted to minimize its neurotoxicity. With this tool, we delineate a wiring diagram between the hippocampus and striatum-two major brain systems for learning, memory, and navigation-that consists of projections from specific hippocampal domains to specific striatal areas via distinct intermediate brain regions. Therefore, this inducible PRVΔIE system provides a tool for dissecting polysynaptic circuits underlying complex brain functions.
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Project description:The attenuated pseudorabies virus (PRV) strain Bartha contains several characterized mutations that affect its virulence and ability to spread through neural circuits. This strain contains a small genomic deletion that abrogates anterograde spread and is widely used as a retrograde-restricted neural circuit tracer. Previous studies showed that the retrograde-directed spread of PRV Bartha is slower than that of wild-type PRV. We used compartmented neuronal cultures to characterize the retrograde defect and identify the genetic basis of the phenotype. PRV Bartha is not impaired in retrograde axonal transport, but transneuronal spread among neurons is diminished. Repair of the U(L)21 locus with wild-type sequence restored efficient transneuronal spread both in vitro and in vivo. It is likely that mutations in the Bartha U(L)21 gene confer defects that affect infectious particle production, causing a delay in spread to presynaptic neurons and amplification of infection. These events manifest as slower kinetics of retrograde viral spread in a neural circuit.
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