New strategy | AAV-assisted RV-ΔG across the blood-brain barrier to achieve whole-brain scale-specific retrograde labeling and cross-multilevel tracing
Time：2024-10-15 16:58:11
“If you want to take something, you must give it to it.” In neural circuit research, neurons precompensate for cell entry receptors to achieve vector retargeting (specific infection) or precompensate for replication-related proteins to achieve neurotropic viral vectors across the synapse. Hitting the mark has become a widely used strategy. For example, in the instrumental transformation of Rabies virus (RV) tracing vector:
(1) RV-EnvA-TVA system: The outer membrane protein EnvA of avian sarcoma virus can specifically recognize the receptor TVA. However, TVA has no endogenous expression in mammalian cells. Compensating TVA in neurons in advance can make EnvA Pseudotyped RV-ΔG achieves specific infection;
(2) RV has the characteristic of retrograde transsynaptic propagation through axon terminal absorption. Glycoprotein-deficient RV (RV-ΔG) loses its transsynaptic ability and can realize RV retrograde by compensating the rabies virus glycoprotein RVG in neurons in advance. Cross-single-level tracing (the next-level neuron has no RVG, so it cannot continue across synapses).
In traditional tracer systems, precompensation of proteins relies on the delivery of a helper virus (usually adeno-associated virus, AAV), but in previous studies, only AAV was usually used for local delivery. In 2017, the Gradinaru experimental team at the California Institute of Technology developed an AAV-PHP.eB mutant for efficient crossing of the blood-brain barrier in C57BL/6 mice, making brain-wide protein compensation possible and also providing tracer tool systems. Development and improvement provide new ideas.
The Xu Fuqiang team of the Chinese Academy of Sciences published an article titled "Brain-wide TVA compensation allows rabies virus to retrograde target cell-type-specific projection neurons" in "Molecular Brain" in early 2022. Based on this, the "AAV- PHP.eB-Assisted Retrograde Tracing Systems (AAV-PHP.eB-Assisted Retrograde Tracing Systems, or PARTS system), and "cell-type-specific PARTS (cPARTS system), through AAV -PHP.eB delivers TVA receptors throughout the brain, achieving whole-brain-scale broad-spectrum retrograde labeling and neuronal cell type-specific retrograde labeling based on RV-EnvA vectors.
Similarly, AAV-PHP.eB was used to deliver RVG throughout the brain, and infect specific brain regions with glycoprotein-deleted rabies virus (RV-ΔG) to achieve reverse cross-multilevel tracing. Furthermore, combined with TVA controlled by a cell type-specific promoter, EnvA-pseudotyped RV-ΔG can be mediated to initiate cell type-specific retrograde trans-polysynaptic tracing. The research paper "Brain-wide N2cG compensation permits glycoprotein-deleted rabies virus to trace neural circuits across multiple synapses" was published in the Journal of Innovative Optical Health Sciences in February 2023.
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“PARTS” & “cPARTS”: New Reverse Marking Strategies
Uptake of viral tracers via neuronal axon terminals and retrograde transport of infected cell bodies can be used to map and manipulate upstream neural networks that project to specific brain regions. Commonly used retrograde labeling tools include N2cG-pseudotyped replication-deficient RV (N2cG-RV-ΔG), AAV2-retro, AAV9-retro, etc. However, AAV2-retro and AAV9-retro have brain region selectivity, while N2cG-RV -ΔG can only achieve broad-spectrum retrograde infection but cannot achieve cell type-specific labeling. New design strategies are urgently needed to solve these problems.
The research team switched to using AAV-PHP.eB that crosses the blood-brain barrier to deliver TVA receptors throughout the brain. Based on this backbone design, on the one hand, it can take advantage of the functional advantages of AAV-PHP.eB across the blood-brain barrier and integrate traditional RV-specific loops. In situ inverse labeling (Figures A and B below) can be extended to whole-brain scale projection network tracking ("PARTS", Figure C below). On the other hand, the Cre-lox system can be used to control TVA components to achieve broad-spectrum retrograde infection. Upgrade to cell type-specific markers (“cPARTS”, Figure D below).
Design Strategies for PARTS/cPARTS Systems
1. Whole-brain compensated TVA to achieve broad-spectrum retrograde labeling of the whole brain
In order to verify whether the AAV-PHP.eB helper virus can effectively mediate retrograde labeling of RV-EnvA, we first tested the designs of PARTS and cPARTS by local injection in the S1→CPu loop. Both PARTS and cPARTS can achieve the traditional labeling system for specific For circuit tracing, the cPARTS system was used to label the population of projection neurons in the S1 brain area that project to the CPu.
Based on the cPARTS system, local injection marks the projection neuron population in the S1 brain area that projects to the CPu.
Further, as a verification of the PARTS system paradigm, AAV-PHP.eB-TVA was delivered to the whole brain through tail vein administration, and RV-EnvA-ΔG-DsRed was injected into the VTA brain area, and multiple upstream brain areas of the VTA were visible. DsRed reporter protein expression includes cerebral cortex (CTX), lateral habenula (LHb), lateral hypothalamic area (LHA), zona incerta (ZI), midbrain reticular nucleus (MRN), and cerebellar nucleus (CBN).
Tracing the broad-spectrum upstream input network of VTA using the PARTS method
2. Combined with whole-brain compensatory TVA in Cre strain mice to achieve whole-brain cell type-specific retrograde tracing
As a verification of the cPARTS system paradigm, Cre-dependent TVA was delivered to the whole brain of VGAT-Cre transgenic mice via AAV-PHP.eB. After injection of RV-EnvA-ΔG-DsRed into the VTA brain area, GABAergic activity in the upstream brain area was marked. Neurons include the globus pallidus lateralis (GPe), lateral hypothalamic area (LHA), zona incerta (ZI), pontine reticular nucleus (PRNr), olfactory tegmental nucleus (LDTg) and vestibular nucleus (VNC), etc.
Tracing GABAergic-specific upstream input networks to the VTA using the cPARTS method
Viral tools available for retrograde tracing across multiple levels of synapses include RV and the Bartha strain of pseudorabies virus (PRV), but PRV cannot infect primates. The development of RV-based multi-level tracing tools faces the following problems: (1) RV wild virus has low safety and can be fatal to animals including humans; (2) As an RNA virus, there is no DNA stage in the virus life cycle and cannot Cell type-specific labeling via Cre-dependent strategies.
Based on this, when the AAV-PHP.eB whole-brain compensation RVG across the blood-brain barrier is used, broad-spectrum reverse cross-multilevel tracing can be achieved (Figure A below). This design takes into account the safety issues of the RV. At the same time, with the use of AAV helper viruses carrying specific promoters, starting cell type-specific reverse cross-multilevel tracing can also be achieved (Figure B below).
To verify the aforementioned design strategy, N2cG was carried by rAAV-PHP.eB and administered intravenously to the whole brain for compensation, and B19G-CVS-N2c-ΔG-EGFP was injected in MOp. Ventral anterolateral thalamus (VAL), basolateral amygdala (BLA), subiculum (SUB), substantia nigra (SNr), simple lobule (SIM), dentate nucleus (DN), and caudate putamen (CPu) Fluorescent signals can be seen in upstream brain areas.
2. Initiating cell type-specific tracing of synapses across multiple levels
To further trace the input network of excitatory neurons in the MOp, AAV9 was used to deliver TVA under the control of the CaMKIIα promoter into the MOp, while rAAV-PHP.eB was injected through the tail vein for whole-brain compensation of N2cG, and 4 weeks later in the MOp Inject EnvA-CVS-ΔG-tdTomato. Many upstream brain areas were labeled by CVS-ΔG-tdTomato, such as the anterior cingulate area (ACA), ventral anterolateral thalamus (VAL), superior colliculus motor-related area (SCm), substantia nigra (SNr), and caudate putamen (CPu) etc.
These results demonstrate that combining EnvA-pseudotyped RV-ΔG with rAAV-PHP.eB carrying a TVA controlled by a cell type-specific promoter can enable initiating cell type-specific tracing of synapses across multiple levels. Based on this principle, researchers can also use Cre-dependent AAV vectors expressing TVA and combine them with Cre transgenic animals to achieve specific labeling.
Initiating cell type-specific tracing of synapses across multiple levels
3. Summary
Among commonly used retrograde tracing tools, AAV2-retro retrograde infection has a brain region preference and is more likely to infect cortical neurons; N2cG-RV-ΔG can only broad-spectrum retrograde label projection networks, and the PARTS/cPARTS strategy overcomes the above shortcomings. On the basis of broad-spectrum labeling, cell type-specific retrograde tracing can be achieved using transgenic animals and conditionally expressed TVA elements carried by AAV-PHP.eB. Many different viral tracers or retrograde tracing methods have been used in the field of neuroscience, and researchers can choose appropriate viral tools based on their experimental needs.
Using blood-brain barrier-crossing rAAV-PHP.eB as a helper virus for whole-brain compensation of RVG, RV-ΔG can be used for reverse trans-polysynaptic tracing. Furthermore, combined with cell type-specific promoters, RV-ΔG pseudotyped with EnvA enables efficient initiation of cell type-specific retrograde tracing across multiple levels of synapses. If this strategy is used for circuit tracing in non-human primates, it is necessary to use AAV serotypes with better cross-blood-brain barrier effects for this species, such as AAV.CAP-B10, AAV.CPP.16, etc.
The PARTS/cPARTS system and the new reverse trans-multi-level synaptic tracing system appropriately graft the advantages of cross-blood-brain barrier AAV vectors into the RV tracing system, enabling the RV-based circuit labeling system to achieve point-to-surface Cross over!
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