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Abstract Neurotropic viral transsynaptic tracing is an increasingly powerful technique for dissecting the structure and function of neural circuits. Herpes simplex virus type 1 strain H129 has been widely used as an anterograde tracer. However, HSV tracers still have several shortcomings, including high toxicity, low sensitivity and non-specific retrograde labeling. Here, we aimed to construct high-brightness HSV anterograde tracers by increasing the expression of exogenous genes carried by H129 viruses. Using a Trojan horse-like strategy, a HSV/AAV (adeno-associated virus) chimaera termed H8 was generated to enhance the expression of a fluorescent marker. In vitro and in vivo assays showed that the exogenous gene was efficiently replicated and amplified by the synergism of the HSV vector and introduced AAV replication system. H8 reporting fluorescence was brighter than that of currently available H129 tracers, and H8 could be used for fast and effective anterograde tracing without additional immunostaining. These results indicated that foreign gene expression in HSV tracers could be enhanced by integrating HSV with AAV replication system. This approach may be useful as a general enhanced expression strategy for HSV-based tracing tools or gene delivery vectors.

Neurotropic viral transsynaptic tracing is an increasingly powerful technique for dissecting the structure and function of neural circuits. Herpes simplex virus type 1 strain H129 has been widely used as an anterograde tracer. However, HSV tracers still have several shortcomings, including high toxicity, low sensitivity and non-specific retrograde labeling. Here, we aimed to construct high-brightness HSV anterograde tracers by increasing the expression of exogenous genes carried by H129 viruses. Using a Trojan horse-like strategy, a HSV/AAV (adeno-associated virus) chimaera termed H8 was generated to enhance the expression of a fluorescent marker. In vitro and in vivo assays showed that the exogenous gene was efficiently replicated and amplified by the synergism of the HSV vector and introduced AAV replication system. H8 reporting fluorescence was brighter than that of currently available H129 tracers, and H8 could be used for fast and effective anterograde tracing without additional immunostaining. These results indicated that foreign gene expression in HSV tracers could be enhanced by integrating HSV with AAV replication system. This approach may be useful as a general enhanced expression strategy for HSV-based tracing tools or gene delivery vectors. [ABSTRACT FROM AUTHOR]

Abstract Background Herpes 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. Methods Based 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. Results H129-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. Conclusions With 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.

The lateral septal nucleus (LS) plays a critical role in emotionality, social behavior and feeding processes, through neural connections with the hippocampus and hypothalamus. We investigated the neural circuits of LS by using herpes simplex virus 1 strain H129 (H129) and pseudorabies virus stain Bartha (PRV). Virus H129 indicates that LS directly projects to some cerebral nuclei (nucleus accumbens, bed nuclei of the stria terminalis and amygdala), part of the hypothalamus (median preoptic, paraventricular, dorsomedial nucleus and lateral area), thalamus (medial habenula, the paraventricular, parataenial and reuniens nuclei, and the medial line nuclei) and the pontine central gray. Then the LS has secondary projections to the CA3 and CA1 field of the hippocampal formation, lateral and medial preoptic area, and the mammillary body. PRV tracing shows that LS are mainly receiving primary inputs from the amygdala, hippocampus, hypothalamic, thalamus, midbrain and hindbrain, and secondary inputs from dorsal and central linear nucleus raphe, the lateral part of the superior central nucleus raphe, the ventral anterior-lateral complex, the intermediodorsal nucleus, the central medial nucleus, the rhomboid nucleus, and the submedial nucleus of the thalamus. The neural circuit data revealed here could help to understand and further research on the function of LS.

• H129 and McIntyre strains spread in neurons and can label neural circuits. • Infection with H129 recombinants decreased PSD-95 reactivity. • Dual infection with H129 and McIntyre recombinants abolished PSD-95 reactivity. • Infection with these recombinant strains caused neurodegeneration. • Infection with these recombinants limits functional study of virally-labeled cells. Neuroinvasive herpes simplex-1 (HSV-1) isolates including H129 and McIntyre cross at or near synapses labeling higher-order neurons directly connected to infected cells. H129 spreads predominately in the anterograde direction while McIntyre strains spread only in the retrograde direction. However, it is unknown if neurons are functional once infected with derivatives of H129 or McIntyre. We describe a previously unpublished HSV-1 recombinant derived from H129 (HSV-373) expressing mCherry fluorescent reporters and one new McIntyre recombinant (HSV-780) expressing the mCherry fluorophore and demonstrate how infections affect neuron viability. Each recombinant virus behaved similarly and spread to the target 4 days post-infection. We tested H129 recombinant infected neurons for neurodegeneration using Fluoro-jade C and found them to be necrotic as a result of viral infection. We performed dual inoculations with both HSV-772 and HSV-780 to identify cells comprising both the anterograde pathway and the retrograde pathway, respectively, of our circuit of study. We examined the presence of postsynaptic marker PSD-95, which plays a role in synaptic plasticity, in HSV-772 infected and in dual-infected rats (HSV-772 and HSV-780). PSD-95 reactivity decreased in HSV-772-infected neurons and dual-infected tissue had no PSD-95 reactivity. Infection by these new recombinant viruses traced the circuit of interest but functional studies of the cells comprising the pathway were not possible because viral-infected neurons died as a result of necrosis or were stripped of PSD-95 by the time the viral labels reached the target. [ABSTRACT FROM AUTHOR]

• H129 anterograde tracers could efficiently label upstream innervating neurons through axon terminal uptake. • For rigorousness, effective modification strategies are needed to avoid or block the axon terminal invasion process of H129. • Attenuated, TK and γ34.5 deleted HSV has the potential to be developed as a useful viral vector for transgene delivery. Herpes simplex virus type 1 H129 strain has been widely used as a useful anterograde neuronal circuit tracing tool. However, whether H129 is a rigorous anterograde tracer and undergoes anterograde-only spreading are questions of significant interest. In the present study, we evaluated the retrograde labeling efficiency of H129 using a TK and ICP34.5 dual deleted H129 recombinant (named as H306) which was replication-deficient in non-dividing postmitotic neurons. The novel tracer was tested in vitro and in vivo for evaluating its invasion properties and tracing capacities. The results demonstrated that H306 could efficiently label the neurons following intracerebral injection. Notably, H306 could also efficiently infect upstream innervating neurons through axon terminal uptake and displayed obvious retrograde labeling phenotype, regardless of 3 days or 10 days of tracing. The data implied that replication-competent, trans-multisynaptic H129 tracing results might be a mixed neural networks from two types of starter cells, because the retrogradely infected neurons would also replicate H129 and spread virus anterogradely through their axon collaterals (ectopic starter sites), as the local infected neurons in the injection site (true starter site). Therefore, the interpretation of the anterogradely tracing neural networks by current H129 tools at longer post-inoculation intervals need to be cautious, and effective modification strategies are needed to avoid or block the axon terminal invasion process of H129, which is important for rigorous anterograde H129 tracer. [ABSTRACT FROM AUTHOR]

The lateral septal nucleus (LS) plays a critical role in emotionality, social behavior and feeding processes, through neural connections with the hippocampus and hypothalamus. We investigated the neural circuits of LS by using herpes simplex virus 1 strain H129 (H129) and pseudorabies virus stain Bartha (PRV). Virus H129 indicates that LS directly projects to some cerebral nuclei (nucleus accumbens, bed nuclei of the stria terminalis and amygdala), part of the hypothalamus (median preoptic, paraventricular, dorsomedial nucleus and lateral area), thalamus (medial habenula, the paraventricular, parataenial and reuniens nuclei, and the medial line nuclei) and the pontine central gray. Then the LS has secondary projections to the CA3 and CA1 field of the hippocampal formation, lateral and medial preoptic area, and the mammillary body. PRV tracing shows that LS are mainly receiving primary inputs from the amygdala, hippocampus, hypothalamic, thalamus, midbrain and hindbrain, and secondary inputs from dorsal and central linear nucleus raphe, the lateral part of the superior central nucleus raphe, the ventral anterior-lateral complex, the intermediodorsal nucleus, the central medial nucleus, the rhomboid nucleus, and the submedial nucleus of the thalamus. The neural circuit data revealed here could help to understand and further research on the function of LS., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Background: Herpes 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., Methods: Based 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., Results: H129-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., Conclusions: With 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.