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An HSV-1-H129 amplicon tracer system for rapid and efficient monosynaptic anterograde neural circuit tracing.

ABSTRACT: Monosynaptic viral tracers are essential tools for dissecting neuronal connectomes and for targeted delivery of molecular sensors and effectors. Viral toxicity and complex multi-injection protocols are major limiting application barriers. To overcome these barriers, we developed an anterograde monosynaptic H129Amp tracer system based on HSV-1 strain H129. The H129Amp tracer system consists of two components: an H129-dTK-T2-pacFlox helper which assists H129Amp tracer's propagation and transneuronal monosynaptic transmission. The shared viral features of tracer/helper allow for simultaneous single-injection and subsequent high expression efficiency from multiple-copy of expression cassettes in H129Amp tracer. These improvements of H129Amp tracer system shorten experiment duration from 28-day to 5-day for fast-bright monosynaptic tracing. The lack of toxic viral genes in the H129Amp tracer minimizes toxicity in postsynaptic neurons, thus offering the potential for functional anterograde mapping and long-term tracer delivery of genetic payloads. The H129Amp tracer system is a powerful tracing tool for revealing neuronal connectomes.

SUBMITTER: Xiong F

PROVIDER: S-EPMC9741617 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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An HSV-1-H129 amplicon tracer system for rapid and efficient monosynaptic anterograde neural circuit tracing.

Xiong Feng F Yang Hong H Song Yi-Ge YG Qin Hai-Bin HB Zhang Qing-Yang QY Huang Xian X Jing Wei W Deng Manfei M Liu Yang Y Liu Zhixiang Z Shen Yin Y Han Yunyun Y Lu Youming Y Xu Xiangmin X Holmes Todd C TC Luo Minmin M Zhao Fei F Luo Min-Hua MH Zeng Wen-Bo WB

Nature communications 20221210 1

Monosynaptic viral tracers are essential tools for dissecting neuronal connectomes and for targeted delivery of molecular sensors and effectors. Viral toxicity and complex multi-injection protocols are major limiting application barriers. To overcome these barriers, we developed an anterograde monosynaptic H129Amp tracer system based on HSV-1 strain H129. The H129Amp tracer system consists of two components: an H129-dTK-T2-pacFlox helper which assists H129Am ...[more]

PMID: 36496505

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Project description:BackgroundViral tracers are important tools for mapping brain connectomes. The feature of predominant anterograde transneuronal transmission offers herpes simplex virus-1 (HSV-1) strain H129 (HSV1-H129) as a promising candidate to be developed as anterograde viral tracers. In our earlier studies, we developed H129-derived anterograde polysynaptic tracers and TK deficient (H129-dTK) monosynaptic tracers. However, their broad application is limited by some intrinsic drawbacks of the H129-dTK tracers, such as low labeling intensity due to TK deficiency and potential retrograde labeling caused by axon terminal invasion. The glycoprotein K (gK) of HSV-1 plays important roles in virus entry, egress, and virus-induced cell fusion. Its deficiency severely disables virus egress and spread, while only slightly limits viral genome replication and expression of viral proteins. Therefore, we created a novel H129-derived anterograde monosynaptic tracer (H129-dgK) by targeting gK, which overcomes the limitations of H129-dTK.MethodsUsing our established platform and pipeline for developing viral tracers, we generated a novel tracer by deleting the gK gene from the H129-G4. The gK-deleted virus (H129-dgK-G4) was reconstituted and propagated in the Vero cell expressing wildtype H129 gK (gKwt) or the mutant gK (gKmut, A40V, C82S, M223I, L224V, V309M), respectively. Then the obtained viral tracers of gKmut pseudotyped and gKwt coated H129-dgK-G4 were tested in vitro and in vivo to characterize their tracing properties.ResultsH129-dgK-G4 expresses high levels of fluorescent proteins, eliminating the requirement of immunostaining for imaging detection. Compared to the TK deficient monosynaptic tracer H129-dTK-G4, H129-dgK-G4 labeled neurons with 1.76-fold stronger fluorescence intensity, and visualized 2.00-fold more postsynaptic neurons in the downstream brain regions. gKmut pseudotyping leads to a 77% decrease in retrograde labeling by reducing axon terminal invasion, and thus dramatically improves the anterograde-specific tracing of H129-dgK-G4. In addition, assisted by the AAV helper trans-complementarily expressing gKwt, H129-dgK-G4 allows for mapping monosynaptic connections and quantifying the circuit connectivity difference in the Alzheimer's disease and control mouse brains.ConclusionsgKmut pseudotyped H129-dgK-G4, a novel anterograde monosynaptic tracer, overcomes the limitations of H129-dTK tracers, and demonstrates desirable features of strong labeling intensity, high tracing efficiency, and improved anterograde specificity.

Project description:BackgroundHerpes simplex virus type 1 strain 129 (H129) has represented a promising anterograde neuronal circuit tracing tool, which complements the existing retrograde tracers. However, the current H129 derived tracers are multisynaptic, neither bright enough to label the details of neurons nor capable of determining direct projection targets as monosynaptic tracer.MethodsBased on the bacterial artificial chromosome of H129, we have generated a serial of recombinant viruses for neuronal circuit tracing. Among them, H129-G4 was obtained by inserting binary tandemly connected GFP cassettes into the H129 genome, and H129-ΔTK-tdT was obtained by deleting the thymidine kinase (TK) gene and adding tdTomato coding gene to the H129 genome. Then the obtained viral tracers were tested in vitro and in vivo for the tracing capacity.ResultsH129-G4 is capable of transmitting through multiple synapses, labeling the neurons by green florescent protein, and visualizing the morphological details of the labeled neurons. H129-ΔTK-tdT neither replicates nor spreads in neurons alone, but transmits to and labels the postsynaptic neurons with tdTomato in the presence of complementary expressed TK from a helper virus. H129-ΔTK-tdT is also capable to map the direct projectome of the specific neuron type in the given brain regions in Cre transgenic mice. In the tested brain regions where circuits are well known, the H129-ΔTK-tdT tracing patterns are consistent with the previous results.ConclusionsWith the assistance of the helper virus complimentarily expressing TK, H129-ΔTK-tdT replicates in the initially infected neuron, transmits anterogradely through one synapse, and labeled the postsynaptic neurons with tdTomato. The H129-ΔTK-tdT anterograde monosynaptic tracing system offers a useful tool for mapping the direct output in neuronal circuitry. H129-G4 is an anterograde multisynaptic tracer with a labeling signal strong enough to display the details of neuron morphology.

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.

Dataset Information

An HSV-1-H129 amplicon tracer system for rapid and efficient monosynaptic anterograde neural circuit tracing.

Publications

An HSV-1-H129 amplicon tracer system for rapid and efficient monosynaptic anterograde neural circuit tracing.

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