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Combination of Neural Loop Tracing and Tissue Transparency Technology Enables Neuroscience Research

The human brain contains approximately one hundred billion neurons, which are interconnected by approximately one hundred trillion synaptic connections, forming an almost infinite number of neural loops. These loops are the structural foundation that supports a variety of basic and advanced brain functions. A deeper understanding of neural loops is essential for understanding the principles of brain function and the pathogenesis of neurological diseases.

Neurons receive information from upstream neurons via dendritic ridges (postsynaptic structures) and send signals to downstream neurons via axon terminals (presynaptic structures). The number of direct upstream and downstream partners per neuron varies widely, from a few hundred to 100,000, and neuronal information can be transmitted through confluent or differentiated neural pathways with monosynaptic or polysynaptic connections.

Neural tracer techniques can map how these neurons are connected to each other. Typically, a paracrine tracer will travel from the cell body to the downstream region of the projection via paraxoplasmic transport, whereas a retrograde tracer can move from the axon terminal to the cell body via retrograde axoplasmic transport. Taking advantage of the natural properties of viral propagation, more and more viruses are being adapted for tracing neural circuits, thus greatly facilitating the study of neural circuits and specific neural projection networks in the brain.

Transneuronal or trans-synaptic viral tracing. (Xu, Xiangmin, et al., 2020)Fig. 1 Schematic illustrations of transneuronal or trans-synaptic viral tracing.1

We present an article that describes specific viral vectors for neuroscience research and details advances in the application of viral vectors for axonal tracing and trans-synaptic tracing. As a partner, Creative Biolabs provides cis- and retrograde tracers for neural loop studies and offers the following related services to help accelerate the progress of your program.

Our Services Descriptions
Viral Vectors Creative Biolabs has long-term devoted to the development and application of various viral vectors for neuroscience research. Based on our advanced platforms and extensive experience, we have finished a lot of challenging work.
Neuronal Plasticity Assay Creative Biolabs provides a range of customized assays to evaluate the neuronal plasticity, which mainly includes but are not limited to Neurogenesis Assay, Synaptogenesis Assay, Neurite Outgrowth/Degeneration Assay, Synaptic Imaging Assay.
Brainbow™ Neuronal Circuit Multicolor Labeling We provide neuronal circuit multicolor labeling toolbox, which allows the visualization and analysis of complex neuronal circuits in the brain. This technology provides a powerful tool for neuroscientists to better understand the function and dysfunction of the brain.

Viruses as Tools for Neural Loop Tracing

Viral tools in neuroscience provide more precise tracing and mapping, and viruses can be localized to specific cell types through genetic strategies to improve transduction efficiency and extend or limit their virulophilicity.

Viruses used in neuroscience research are typically genetically modified or recombinant strains of wild-type viruses, including:

Virus Type Descriptions Advantages Disadvantages
Adeno-associated virus (AAV) AAV is a single-stranded positive-sense DNA genome with approximately 5 kb base pairs. Recombinant AAV (rAAV), developed from wild-type AAV, is one of the most commonly used viral tools in biomedicine. rAAV is often used in conjunction with Cre/loxP and flippase (Flp)/flippase-recognition-target (FRT) recombinase technologies, which are generally applicable to all DNA viruses.
  • Excellent safety profile
  • Low immunogenicity
  • Multiple serotypes
  • Wide range of infected host cells
  • Ability to mediate long-term stable expression of exogenous genes in vivo
  • Smaller transgene volume (~4.8 kb)
Pseudorabies virus (PRV) PRV is a member of the subfamily α-herpesviruses, with a diameter of 200-250 nm and a genome of about 150 kb. It is a spherical double-stranded DNA virus with an envelope that allows insertion of large exogenous genes. Its strains include Becker and Bartha; the Bartha strain is less virulent, has a high retrograde transmission capacity, and is widely used in neurocircuit studies. Widespread use in neural loop studies
  • Recombinant PRVs such as PRV531 and PRV724 have robust fluorescent labeling and can be effectively used as retrograde transmultisynaptic tracers.
  • Recombinant PRV Ba2017 and PRV-hSyn-Cre are designed to target gene expression and facilitate functional dissection of neural circuits in vivo.
  • Risk of infecting the human central nervous system and causing encephalitis
Rabies virus (RV) RV is an enveloped, negative-stranded, single-stranded RNA virus. RV is highly neuroaffinity and replicates by means of a self-encoding RNA polymerase complex. rv-ΔG is a genetically modified rabies virus with the envelope glycoprotein gene deleted, and is commonly used as a neuroscience research RV-ΔG is a genetically modified rabies virus with the envelope glycoprotein gene deleted and is commonly used as a tracking tool in neuroscience research.
  • Can target infection to specific neurons
  • Allow for single synapse tracking studies
  • Infection is relatively inefficient
  • Higher virulence
HSV1 strain H129 HSV1 strain H129 was originally isolated from the brains of patients with acute HSV encephalitis and has been widely used as an output network tracer because of its property of trans-synaptic propagation in a paracrine manner.
  • High replication capacity
  • Transmultisynaptic
  • Low labeling brightness
  • Non-specific retrograde labeling
  • Toxicity
  • Require immunostaining to amplify the fluorescent signal to visualize the output neural loop
Vesicular stomatitis virus (VSV) VSV is a bullet-shaped enveloped negative-stranded RNA virus. It is capable of paracrine transmission between neurons and can be pseudotyped by pretending that its G-protein is pseudotyped with other viral glycoproteins with different marking properties and synaptic transmission types.
  • Infect most mammalian cells
  • Rapid replication
  • Efficient infection
  • Gene expression capability
  • Suitable for downstream recursive tracking of neural networks
  • High virulence
  • Rapidly lethal
Lentivirus (LV) LV is a spherical enveloped virus with a diameter of about 80-100 nm. Human immunodeficiency virus type 1 (HIV-1)-based LV vectors are the most widely used in neuroscience research and are capable of stable and efficient in vivo gene transfer to mature neurons and infection of dividing and nondividing cells, including neurons and glial cells in the adult mammalian brain.
  • High packaging capacity
  • Inefficient retrograde gene transfer

A number of tracer products are important tools commonly used in research. You can browse the table below to see a list of our recommended products.

Cat. No Product Name Types
NTA-2011-ZP11 PRV-CMV-EGFP Retrograde,multisynaptic
NTA-2011-ZP12 PRV-CMV-RFP Retrograde,multisynaptic
NTA-2011-ZP13 PRV-hUbC-EGFP Retrograde,multisynaptic
NTA-2011-ZP15 HSV-EGFP Anterograde,multisynaptic
NTA-2011-ZP16 HSV-tdTomato Anterograde,multisynaptic
NTA-2011-ZP17 HSV-LSL-tdtomato-2aTK(H356) Credependent,Anterograde,multisynaptic
NTA-2011-ZP20 VSV-eGFP Anterograde,multisynaptic
NTA-2011-ZP21 VSV-mCherry Anterograde,multisynaptic
NTA-2011-ZP22 VSV-BFP Anterograde,multisynaptic

Application Cases of Tissue Transparency Techniques in Neural Loop Studies

Choosing the right tracer/tracer virus to accomplish neural labeling efficiently is the basis for probing neural loop studies. Next, optical imaging techniques are needed to detect the projections of the entire loop. Traditional tissue slicing methods allow precise brain localization of neuronal signals in the projection area, but the information at the level of two-dimensional slices does not provide a complete picture of the transmission and projection of nerve fibers. In recent years, tissue transparency techniques have been rapidly developed to reduce light attenuation in biological tissues by equalizing the refractive indices between different cellular components and removing pigments to improve the imaging depth, providing an effective tool for 3D imaging of thick tissues or even whole organisms.

  • 3D Visualization of Peripheral Nerve Circuits - The researchers first injected fluorescently labeled AAV virus at the dorsal root ganglion (DRG) of the mouse spinal cord, where the virus delivered a tracer labeling the downstream nerves in passing, and combined it with tissue transparency and light-sheet micrographic imaging to better trace the pathways of sensory neurons that extend into adipose tissue.
  • 3D Visualization of Central Nervous Circuits - Using viral tracer technology, scientists injected AAV-eGFP and AAV-tdtomato viruses in the sweet-taste-responsive cortex (GCsw) and bitter-taste-responsive cortex (GCbt), respectively, and, in conjunction with tissue-transparency and light-sheet micrographic imaging systems, found that neurons in the sweet-taste-responsive and bitter-taste-responsive cortices projected to different regions in the amygdala.
  • 3D Visualization of Peripheral-Central Neural Circuits - Investigators meta-injected pseudorabies virus PRV152-EGFP into the superficial flexor digitorum profundus muscle, where the virus was retrogradely delivered from within the peripheral skeletal muscle to the lower motor neurons in the spinal cord and completed trans-synaptic transmission to illuminate the upper motor neurons in the brain, and then subsequently, using the FDISCO Transparencies method and light-sheet microscopy, the functional connectivity of functional connections that regulate the motor function of skeletal muscle in the brain were neurons were imaged.

Creative Biolabs provides various types of neural tracer markers as well as a number of leading-edge technologies to solve the challenges encountered in neural tracer loop research, and help research in the field of brain science and neuroscience.


  1. Xu, Xiangmin, et al. "Viral vectors for neural circuit mapping and recent advances in trans-synaptic anterograde tracers." Neuron 107.6 (2020): 1029-1047.
For Research Use Only. Not For Clinical Use.
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