Neural Circuit Tracing: Tools
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TO PROMOTE NEUROSCIENCE RESEARCH CONTRIBUTE TO BUILD A HEALTHIER TOMORROW CONTENTS
01 Neuroscience and Virus Vector
Viral Vectors Tools for Neuroscience Types of Viral Vector Tools Anterograde/Retrograde Transfer of Viruses
02 Virus Vector-based Gene Regulation
Principles of Gene Regulation Tissue/Cell-specific Gene Regulation
03 Virus Vector in Neural Circuit Tracing
Neural Circuit Tracing Technology Common Neural Circuit Tracing Tools
Page 03 01 Neuroscience and Virus Vector
Viral Vectors for Neuroscience
Molecular Level Cell Level Circuit Level Behavior Level
Types of Viral Vectors
Adeno-associated Virus (AAV) Retro/Lentiviruses (Rv/Lv) Rabies Virus (RABV) Herpes Simplex Virus (HSV) Pseudorabies Virus (PRV)
Anterograde/Retrograde Transfer of Viruses
Neural Circuit Tracing Tools Page 04 01 Neuroscience and Virus Vector OUR MISSION
Innovating healthcare with Creative Biolabs
Everything we do is rooted in neuroscience. Creative Biolabs, part of Creative Biolabs, is a biotechnology company that focuses on discovering, developing, manufacturing, and delivering innovative and high-quality scientific products for worldwide neuroscientists.
Neural Circuit Tracing Tools Page 05 01 Neuroscience and Virus Vector VIRAL VECTORS FOR NEUROSCIENCE
Neuroscience research can be divided into four levels: molecules, cells, circuits, and behaviors.
l Molecular Level
Mainly explore the functions of different genes and their coded proteins in neurons. Through gene expression control technology, we can change the expression of target genes in cells and determine gene function in combination with disease or cell phenotype changes. Commonly used techniques include overexpression, interference, and knockout.
l Cell Level
The main purpose is to find neurons related to different diseases or behaviors, and further study their functions. Generally, calcium ion imaging technology is used to identify related neurons, and then optogenetic, chemical genetics, and other techniques are used to manipulate neuronal activities to study their functions.
Neuroscience and Virus Vector Page 06 01 Viral Vectors For Neuroscience l Circuit Level
After determining the function of a neuron, we must further study the coordinated regulation function between the neurons, as well as the respective roles played by the circuits connected to different neurons. It is often necessary to trace the neural circuit and manipulate the activity of the neural circuit to study its function. Common techniques include neural circuit tracing, optogenetics, and chemogenetics.
l Behavior Level
Mainly detect the changes in the behavior of different behavior models after the regulation of molecules, cells, and circuit. The most important thing in behavioral level research is to choose an appropriate animal behavior paradigm.
From molecular engineered, cell-type specific viruses, to optogenetics and chemogenetics, virus vectors continue to evolve rapidly and to transform the field of behavioral and functional neuroscience. Viral vectors expand the neurobiology toolbox to include direct and rapid anterograde, retrograde, and trans-synaptic delivery of tracers, sensors, and actuators to the mammalian brain.
Neuroscience and Virus Vector Page 07 01 Viral Vectors For Neuroscience TYPES OF VIRAL VECTORS
Recombinant viruses are the workhorse of modern neuroscience. They are now used routinely to study the molecular and cellular functions of a gene within an identified cell type in the brain, and enable the application of optogenetics, chemogenetics, calcium imaging, and related approaches. Three of the most common types of viruses used for gene delivery in neurobiology are adeno-associated viruses (AAVs), retro and lentiviruses, and glycoprotein deleted rabies virus (Rabies dG).
l Adeno-associated Virus
AAV has been proved as the most excellent gene therapy vector. To date, more than 204 clinical trials have been carried out using AAV vectors for gene delivery. The introduction of the AAV in neuroscience has helped advance both circuit identification and functional circuit analysis, and the result has dramatically expanded the capabilities of neuronal circuit characterization.
Neuroscience and Virus Vector Page 08 01 Types of Viral Vectors rAAV Serotype
Over the past decades, numerous AAV serotypes have been identified with variable tropism. Genome divergence among different serotypes is most concentrated on hypervariable regions (HVRs) of virus capsid, which might determine their tissue tropism. To effectively enable the transduction of tissues or limit AAV tropism to specific tissues in vivo, researchers should choose the appropriate serotype according to the purpose of each experiment.
Advantages and Disadvantages of AAV
Neuroscience and Virus Vector Page 09 01 Types of Viral Vectors l Retro/Lentiviruses
Viruses of the Retroviridae or Retrovirus family, including gamma- retrovirus and lentivirus genera, have the unique ability to integrate permanently into the host genome and thereby enable long-term stable gene expression. Gamma-retroviral vectors are derived from the Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV) or Murine Stem Cell Virus (MSCV) genomes whereas lentiviral vectors are derived from the human immunodeficiency virus (HIV) genome.
Gamma-Retrovrial vs. Lentiviral Vector Systems Gamma-retroviruses (often referred to as retroviruses) cannot penetrate the nuclear envelope and only transduce cells during division when the nuclear envelope breaks apart.
Neuroscience and Virus Vector Page 10 01 Types of Viral Vectors Since lentiviruses trigger a lessened immune response compared to retroviruses and are capable of infecting both dividing and non-dividing cells, the use of recombinant lentiviruses is more prevalent for the delivery of genes to non-dividing cells such as neurons. Lentiviruses are not as efficient as AAVs for in vivo gene delivery and their delivered genes are expressed less robustly.
Recombinant retro/lentiviruses are also designed to be infectious but non-propagating. During the development of recombinant lentiviral vectors, the viral genome was divided among multiple plasmids, and the machinery necessary for viral replication was removed from the viral particles. Each subsequent generation of lentiviral vectors was designed to enhance safety features for use in research laboratories.
Advantages and Limitations of Retro/Lentiviruses
Neuroscience and Virus Vector Page 11 01 Types of Viral Vectors l Rabies Virus (RABV)
To understand the underlying architecture of the brain, researchers often rely on fluorescent neural tracers delivered by glycoprotein deleted Rabies (Rabies dG) viruses. Currently, rabies strains SADB19 and CVS- N2c genomes have been reconstituted by recombinant technologies and their transfer vectors can deliver 8.5 kb of genetic material. Rabies dG infects neurons and spreads trans-synaptically in a retrograde direction to identify connecting neurons and map signaling pathways in neural circuits. Recombinant technology has allowed researchers to modify rabies dG to create safer alternatives.
Rabies G protein plays a predominant role in retrograde transfer of virus among neural circuitry. When used for pseudotyping, rabies G promotes retrograde transmission of other types of enveloped proteins such as lentiviruses as well. Rabies G is the sole exposed protein on the surface of the virus and is necessary for the trans-synaptic transfer of the virus.
Features of Rabies Virus
Infection of neuronal cells
Trans-synaptic dissemination
Transient transgene expression, not integrating genome
About 3-5 kb DNA uptake capacity
ss (-) RNA genome
Bullet shaped. 250 nm length, 80nm diameter
Security level S1 (if G-protein deleted)
Neuroscience and Virus Vector Page 12 01 Types of Viral Vectors RABV as a Monosynaptic Tracer
The RABV has a single-stranded RNA genome of negative polarity of about 12 kb. In the cDNA of the SAD-B19 strain, the glycoprotein gene was completely removed (G-deleted). Since transsynaptic transfer is only possible in the presence of the G protein, it must be provided in trans (by the first injection with AAVs, LVs, or by transgenic G protein-expressing animals). Thus, for infection and subsequent trans-synaptic transfer, the receptor and the G protein must first be provided. Instead of the G protein, other proteins, such as eGFP, can be expressed by the RABV. The figure below describes the essential steps.
Neuroscience and Virus Vector Page 13 01 Types of Viral Vectors l Herpes Simplex Virus (HSV)
Herpes simplex virus (HSV) is a prevalent neurotropic virus, which establishes lifelong latent infections in the neurons of sensory ganglia.
HSV has been widely used as an anterograde tracer. After adding the modified HSV with fluorescent protein, it can be used not only to efficiently label the connections between different brain regions, but also can label the connections between peripheral and central.
Pseudorabies virus as an anterograde tracer
After replicating in surface epithelia, herpes simplex virus type-1 (HSV-1) enters the axonal terminals of peripheral neurons. The viral genome translocates to the nucleus, where it establishes a specialized infection known as latency, re-emerging periodically to seed new infections. VP16 is required for productive replication in neurons, and thus the absence of tegument-derived VP16 facilitates establishment of latency.
Neuroscience and Virus Vector Page 14 01 Types of Viral Vectors Reactivation stimuli can elicit many changes in the neuron, including nuclear accumulation of HCF-1 and VP16, which is synthesized de novo along with other viral regulatory proteins. Stimulation of viral lytic transcription by VP16 leads to viral DNA amplification and synthesis of virion proteins. Capsids are transported in an anterograde fashion to the axonal termini where they mature and are then released, bringing the HSV-1 life cycle full circle.
Neuroscience and Virus Vector Page 15 01 Types of Viral Vectors Anterograde/Retrograde Transfer of Viruses
Creative Biolabs offers AAV, RV/LV, VSV, PRV, RABV, HSV, etc., to promote neuronal circuit tracing studies. The table below provides a brief overview of the properties of different types of viruses.
Type Virus name Classification Genomic type
Single stranded Adeno-associated virus, AAV Parvoviridae DNA
Double strands Canine adenovirus, CAV Adenovirus DNA
+ Single stranded Non- transsynaptic Semliki Forest virus, SFV Togaviridae RNA
Rabies virus (Glycoprotein G- -Single stranded Rhabdoviridae deleted), RV-∆G RNA
Herpes simplex virus Double strands Herpesviridae amplicon, HSV amplicon DNA
Herpes simplex virus, HSV Double-stranded Herpesviridae H129 DNA Anterograde, multi-synaptic -Single stranded Vesicularstomatitis virus, VSV Rhabdoviridae, RNA Trans-multi-synaptic Double-stranded Pseudorabies virus, PRV Herpesviridae, RNA Retrograde, multi-synaptic -Single stranded Rabies virus,RV WT Rhabdoviridae, RNA Trans-synaptic Herpes simplex virus (TK- Double-stranded Herpesviridae, deleted), HSV-∆TK DNA Anterograde, monosynaptic Adeno-associated Single stranded Parvoviridae, virus,serotype 1, AAV1 DNA Trans-monosynaptic -Single stranded Rabies virus, RV-∆G-EnvA Rhabdoviridae, RNA Retrograde, monosynaptic Pseudorabies virus (TK- Double-stranded Herpesviridae deleted), PRV-∆TK RNA
In addition to the listed viruses above, we also provide pre-made and custom virus services. Our highly experienced scientists are always ready to work with you.
Neuroscience and Virus Vector Page 16 01 Anterograde/Retrograde Transfer of Viruses l Pseudorabies Virus (PRV)
Pseudorabies virus (PRV), an α-herpesvirus with broad host range, reveals chains of functionally connected neurons in the nervous systems of a variety of mammals.
PRV has a double-stranded DNA genome, and the genome is approximately 150 kb that encodes capsid, tegument, and envelope protein. The retrograde trans-synaptic PRV is modified from vaccine strain Bartha, which can be used to analyzePseudorabies thevirus as a structuretrans-neuronal tracer of neuronal connectivity in brain center and periphery. After the virus infects nerve cells, it can replicate and express the target gene in the cell. The progeny viruses are transported to the synapse and then retrograde trans- synaptic labeling of the upstream neuron for further replication and transmission across the synapse.
Neuroscience and Virus Vector Page 17 01 Anterograde/Retrograde Transfer of Viruses Pseudorabies virus as a trans-neuronal tracer
Figure. Trans-synaptic spread of PRV infection in a neural circuit. (a) After infecting primary neurons (shown in green), wild-type strains of PRV, such as PRV-Becker or PRV-Kaplan, promote infection that spreads from the cell body to axon terminals of pre-synaptic neurons (retrograde spread). Virions of wild-type strains might also be transported down the axon to infect the cell bodies of post-synaptic neurons (anterograde spread). (b) PRV-Bartha replicates well in neurons, but infected animals have reduced symptoms compared to wild-type virus infections. In addition, PRV- Bartha infection spreads only from post- to pre-synaptic neurons in a circuit. Infection by PRV-Bartha cannot spread from pre- to post-synaptic neurons because structural components of virions cannot be sorted into axons, a process that requires the actions of viral proteins gE, gI and US9. The genes encoding these proteins are deleted in the PRV-Bartha genome.
Neuroscience and Virus Vector Page 18 01 Anterograde/Retrograde Transfer of Viruses 02 Virus Vector-based Gene Regulation
Principles of Gene Regulation
Target gene knockdown-RNA interference Target gene knockout Target gene overexpression
Tissue/Cell-specific Gene Regulation
In situ injection Tissue-specific serotype Tissue-specific promoter Recombinase-dependent expression system
Neural Circuit Tracing Tools Page 19 02 Virus Vector-Based Gene Regulation AAV9 DNA of the Vector virus is removed
Figure. Schematic overview of how viruses (i.e. adenovirus) can be used to deliver recombinant genetic material to neurons, or other cell types within the nervous system. Steps include viral binding to cell membrane, endosomal packaging and breakdown, delivery and integration of vector into host DNA, and ending with the expression of the desired protein.
Neural Circuit Tracing Tools Page 20 02 Virus Vector-Based Gene Regulation PRINCIPLES OF GENE REGULATION
l Ta r g e t gene knockdown
Gene knockdown is an experimental technique by which the expression of one or more of an organism's genes is reduced. The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript. RNA interference (RNAi) is a means of silencing genes by way of mRNA degradation. Gene knockdown is achieved by introducing small double- stranded interfering RNAs (siRNA) into the cytoplasm.
Principle
Small interfering RNAs can originate from inside the cell or can be exogenously introduced into the cell. Once introduced into the cell, exogenous siRNAs are processed by the RNA-induced silencing complex (RISC). The siRNA is complementary to the target mRNA to be silenced, and the RISC uses the siRNA as a template for locating the target mRNA. After the RISC localizes to the target mRNA, the RNA is cleaved by a ribonuclease.
Virus Vector-Based Gene Regulation Page 21 02 Principles of Gene Regulation Virus Vector-Based Gene Regulation Page 22 02 Principles of Gene Regulation l Ta r g e t gene knockout
A gene knockout (KO) is a genetic technique in which one of an organism's genes is made inoperative ("knocked out" of the organism).
Principle
A conditional gene knockout allows gene deletion in a tissue in a time specific manner. This is required in place of a gene knockout if the null mutation would lead to embryonic death. This is done by introducing short sequences called loxP sites around the gene. These sequences will be introduced into the germ-line via the same mechanism as a knock-out. This germ-line can then be crossed to another germline containing Cre- recombinase which is a viral enzyme that can recognize these sequences, recombines them, and deletes the gene flanked by these sites.
Figure. Conditional gene knockouts using the Cre recombinase-loxP recombination system.
Virus Vector-Based Gene Regulation Page 23 02 Principles of Gene Regulation l Ta r g e t gene overexpression
Gene overexpression is the process which leads to the abundant target protein expression subsequently.
There are two main strategies to achieve the overexpression of the target gene: introducing the exogenous coding gene and enhancing the transcription level of endogenous gene. Exogenous introduction of coding gene sequences is the most current strategy used in overexpression regulation. The principle is to construct the coding sequence (CDS) of the target gene into a viral vector and introduce it into the cell to express a large amount of the coding sequence of the target gene, so as to increase the expression of the target gene.
Virus Vector-Based Gene Regulation Page 24 02 Principles of Gene Regulation TISSUE/CELL-SPECIFIC GENE REGULATION
l In Situ Injection
In situ injection is a commonly used injection method for virus infection of the nervous system. By using brain stereotactic injection technology, the injected virus can directly transduce into the target brain area, thereby achieving region-specific gene regulation. However, the spread of the virus is limited, infecting only nerve cells near the injection site.
l Tissue-specific Serotype
In situ injection can only ensure the specificity of the injection area. When the target area is large, the area is scattered, or cell-specific infection is required, this method cannot meet the experimental needs. In this case, tissue-specific serotypes should be used.
Take AAV as an example. The intensity of gene expression and tissue tropism depends highly on the AAV serotype used in different tissues. Different serotypes differ mainly in the surface protein (capsid). Carefully choosing the serotype enables robust gene expression in specific tissues with minimal immune response. The table gives an overview of the different serotypes.
Virus Vector-Based Gene Regulation Page 25 02 Tissue/Cell-Specific Gene Regulation Table. Common AAV Serotypes and their target tissues.
In addition to choosing the optimal serotype for specific tissue, more methods have been developed to improve brain transduction after systemic AAV injection, such as virus genome tuning, time of injection, as well as capsid engineering.
Figure. Summary of methods used to improve gene delivery after systemic injection of AAVs.
Virus Vector-Based Gene Regulation Page 26 02 Tissue/Cell-Specific Gene Regulation l Tissue-specific Promoter
Although tissue-specific serotypes can achieve cell specificity, there are very few cell-specific surface receptors, which leads to the application limitations of tissue-specific serotypes. Tissue-specific promoters is an ideal solution to overcome this limitation. The use of specific promoters allows the expression of the gene of interest under specific conditions.
Tissue or cell-specific promoters allow:
The expression of genes of interest into targeted organs or tissues such as liver, brain, lung.
The discrimination or selection of a population of cells within a tissue using fluorescent proteins (i.e., oligodendrocytes versus neurons). To follow the differentiation process of living cells in a tissue.
Virus Vector-Based Gene Regulation Page 27 02 Tissue/Cell-Specific Gene Regulation Unlike broad-spectrum promoters, tissue-specific promoters are generally identified from gene promoters specifically expressed by specific cells, which can regulate the expression of foreign genes in specific cells or tissues. The table below summarizes common tissue- specific promoters in the nervous system.
Promoter Specificity Promoter Specificity
hSyn Neurons gfaABCID Astrocyte
mecp2 Pseudo-unipolar neurons Iba1 Microglia
TUBA1A Sensory neurons F4/F80 Microglia
c-fos Forebrain glutamatergic neurons CD68 Microglia
CaMKIla GABAergic neurons/Interneurons CNP Microglia/Schwann cells
hVGAT Dopamine neuron Cx3cr1 Microglia
Slc6a3 Dopamine neuron GFAP Astrocytes
INA Neuroprogenitors MOBP Oligodendrocytes
MBP Oligodendrocytes TH Dopamine neuron
GFAP Astrocytes NES Neuroprogenitors
FOXA2 Dopamine neuron More Updating
Virus Vector-Based Gene Regulation Page 28 02 Tissue/Cell-Specific Gene Regulation l Recombinase Dependent Expression System
Further regulation of gene expression can be achieved using recombinase (e.g., Cre, Flp, Dre) transgenics and recombinase sensitive sequences in viral transfer vectors (e.g., gene of interest flanked by lox-P sequence). Unlike AAVs, retros, or lentiviruses, the rabies virus is maintained as an RNA-protein complex in the cytoplasm of cells and its genes are expressed via a specialized viral polymerase. Therefore, neuron-specific promoters or recombinase recognition sites cannot be introduced onto recombinant rabies transfer vectors/plasmids.
Principles of Site-specific Recombinase (SSR) Te c h n o l o g y
Site-specific recombinases are proteins that bind to distinct DNA target sequences. Two of the most exciting and versatile genetic tools are the Cre-lox and FLP-FRT technologies. Both allow the location and timing of gene expression to be closely regulated.
The Cre-lox system
Cre recombinase is a Type I topoisomerase that catalyzes site-specific recombination of DNA between two loxP (locus of X-over P1) sites. The Cre/lox system does not require any cofactors. LoxP sequences contain distinct binding sites for Cre recombinases that surround a directional core sequence where recombination and rearrangement take place. When cells contain loxP sites and express the Cre recombinase, a recombination event occurs.
Virus Vector-Based Gene Regulation Page 29 02 Tissue/Cell-Specific Gene Regulation The FLP-FRT system
The FLP-FRT system is similar to the Cre-lox system and is becoming more frequently used in mouse-based research. It involves using flippase (FLP) recombinase, derived from the yeast Saccharomyces cerevisiae. FLP recognizes a pair of FLP recombinase target (FRT) sequences that flank a genomic region of interest.
The use of other site-specific recombination systems in combination with Cre-lox allows for even greater flexibility and control of genetic experiments. For example, one can target tissue-specific gene expression with even more precision by using two tissue-specific promoters – one to express Cre, and the other to express FLP. This approach can be used to express any gene of interest only at the intersection (in space or time) where Cre and FLP co-exist.
Figure. An intersectional switch can be used to express the gene of interest in only one cell type with great specificity.
Virus Vector-Based Gene Regulation Page 30 02 Tissue/Cell-Specific Gene Regulation 03 Virus Vector in Neural Circuit Tracing
Neural Circuit Tracing Technology
Anterograde Tracing
Retrograde Tracing
Anterograde and Retrograde Tracers
Common Neural Circuit Tracing Tools
Fluorescent Neuronal Tracers
Hydrazides & Biocytins
Labeled Dextran Conjugates
Lipophilic Tracers
Protein Tracers
Viral Tracers
Neural Circuit Tracing Tools Page 31 03 Virus Vector In Neural Circuit Tracing A key aspect of understanding the brain's function is knowing its architecture, in particular the connections between different brain regions. Directional flow of information between different brain regions is mediated via individual neurons. A neural circuit is a population of neurons interconnected by synapses to carry out a specific function when activated. Neural circuits interconnect to one another to form large scale brain networks. Therefore, tracing neuronal circuits is necessary for neuroscientists to identify connectivity patterns between components of neural system.
Figure 1: Examples of neuronal circuits.
Neural Circuit Tracing Tools Page 32 03 Virus Vector In Neural Circuit Tracing NEURAL CIRCUIT TRACING TECHNOLOGY
l Anterograde/Retrograde Tracing
Anterograde tracing outlines neurons from their cell bodies to the terminals of their axons, while retrograde tracing outlines neurons in the opposite direction, from the terminals of their axons to their cell bodies.
Anterograde and retrograde tracing take advantage of existing transport pathways in neurons. Anterograde transport is typically used for the trafficking of organelles, such as mitochondria, as well as macromolecules like actin and myosin, and enzymes for transmitter synthesis. Retrograde transport is used to transport endocytosed material or molecules targeted for degradation.
Virus Vector In Neural Circuit Tracing Page 33 03 Neural Circuit Tracing Technology l Anterograde and Retrograde Tracers
Conventional (Mainly) Retrograde Tracers
Methods Products Applications Advantages Drawbacks Ages
Easily diffuse; Responsive; cost- Horseradish Retrograde poor specificity; 1970s CB-HRP effective; long- peroxidase neuron tracing cell death after - lasting signal treatment
Rapid transport (2–7 Cholera toxin Retrograde Limited range 1977s CTb conjugates days), low toxicity; subunit B neuron tracing of applications - ease of use
Fluorescent Highly resistant to Retrograde 1980s latex Retrobeads fading and offer Difficult to use neuron tracing - microspheres long-term labeling
Label small Label live cells and Limited range 1990s Phytoagglutinin WGA; PHA diameter have transsynaptic of applications - sensory neurons capabilities
Fast Blue, Inorganic Diamidino Yellow, Autonomic, Not dependent on Easily diffuse; 2000s fluorescent True Blue and the motor or active transport; poor specificity - tracers carbocyanines DiI sensory neurons used for fixed tissue and DiO
Virus Vector In Neural Circuit Tracing Page 34 03 Neural Circuit Tracing Technology Conventional (Mainly) Anterograde Tracers
Methods Products Applications Advantages Drawbacks Ages
Long storage time; Anterograde Combined with a variety Can only label Particles BDA 1980s- neuron tracing of fluorescein and injured neuron chemical methods
Longer post- Label small Label live cells and have injection survival Phytoagglutinin PHA diameter sensory 1990s- transsynaptic capabilities times (10 to 20 neurons days)
Limitations of Conventional Tracers
The use of conventional tracers are common. However, their major limitations should be considered.
1 Conventional tracers can be taken up by fibers of passage, which can lead to incorrect identification of projections.
2 Many conventional tracers spread around the injection site.
Tracer uptake relies predominantly on sugars that are located 3 on the glycocalyx of most, if not all, neurons, or relies on common mechanisms such as endocytosis.
The direction of axonal transport is rarely exclusive, which 4 complicates circuit analysis.
Virus Vector In Neural Circuit Tracing Page 35 03 Neural Circuit Tracing Technology Viral Tracers
Recombinant viral vectors that drive the expression of fluorescent "reporter" proteins in transduced neurons have been widely adopted by neuroscientists because of their directional specificity, the high (in most cases permanent) levels of reporter expression obtained, and the absence of transduction of fibers of passage.
Viral tracers may be divided into two distinct classes. One is static vectors that remain locked within the targeted cell population and essentially function like conventional tracers, which are usually replication-deficient. The other is the vectors that spread through linked circuits via trans- synaptic travel, which are almost always replication-competent.
AAV9 AAV1 AAV-Retro HSV RV/PRV
Direction Anterograde Anterograde Retrograde Anterograde Retrograde
Monosynaptic/P Monosynaptic/P Trans-synaptic Static Monosynaptic Static olysynaptic olysynaptic
Table: Common Neural Circuit Tracer Virus.
Virus Vector In Neural Circuit Tracing Page 36 03 Neural Circuit Tracing Technology COMMON NEURAL CIRCUIT TRACING TOOLS
l Fluorescent Neuronal Tracers
Fluorescent tracers have the advantage of direct imaging or can be used as haptens for antibodies or streptavidin dye conjugates for signal amplification.
Creative Biolabs' NeuroTrace™ molecular probe labeling and detection tools include multiple probe options for dissecting neural networks and their functions, and each type of probe has an extensive fluorescent color palette. Fast Blue (FB), Diamidino Yellow (DY), True Blue (TB), and Granular Blue (GB) are the most common fluorescent dyes as retrograde neuron tracers.
Common Fluorescent Neuronal Tracers
Cat. No. Product Name Excitation (nm) Emission (nm)
NTA-2011-ZP29 Fast Blue (FB) 365 420
NTA-2011-ZP30 Diamidino Yellow (DY) Diaceturate 365 > 500
NTA-2011-ZP31 True Blue (TB) Diaceturate Salt 365 405
NTA-2011-ZP32 Granular Blue (GB) Dihydrochloride 365 410
Virus Vector In Neural Circuit Tracing Page 37 03 Common Neural Circuit Tracing Tools l Hydrazides & Biocytins
Hydrazides and biocytins are a class of cell tracers that can be fixed by aldehyde. These low molecular weight (<1,000 Da) polar tracers can cross gap junctions and follow neuronal projections. Biocytin hydrazide provides a variety of fluorescent colors and can be used in multiples with other probes. Biocytins are easy to accept secondary detection for effective signal amplification.
Properties of Hydrazides and Biocytins
Hydrazides Biocytins
Tissue sections Yes Yes
In vivo neurons Yes Yes
Fixed tissue sections No No
Cultured cells Yes Yes
Cell loading Microinjection, iontophoresis Microinjection, iontophoresis
Staining mode Endogenous Endogenous
Best for Single cells Endogenous
Transport Bidirectional Bidirectional
Synaptic transfer No No
Gap junction transfer Yes Yes
Virus Vector In Neural Circuit Tracing Page 38 03 Common Neural Circuit Tracing Tools Hydrazides for Neuron Staining
Standard Cat. No. Product Name Fluorophore Ex/Em (nm) filter set(s)
Alexa Fluor® NTA-2011-ZP80 Alexa Fluor® 350 Hydrazide DAPI 346/442 350
Alexa Fluor® NTA-2011-ZP81 Alexa Fluor® 488 Hydrazide FITC 495/519 488
Alexa Fluor® NTA-2011-ZP82 Alexa Fluor® 568 Hydrazide Rhodamine 578/603 568
Alexa Fluor® NTA-2011-ZP83 Alexa Fluor® 594 Hydrazide Texas Red® 590/617 594
Alexa Fluor® NTA-2011-ZP84 Alexa Fluor® 555 Hydrazide TRITC 555/565 555
Alexa Fluor® NTA-2011-ZP85 Alexa Fluor® 633 Hydrazide Texas Red® 632/647 633
Alexa Fluor® NTA-2011-ZP86 Alexa Fluor® 647 Hydrazide Cy®5 650/668 647
Virus Vector In Neural Circuit Tracing Page 39 03 Common Neural Circuit Tracing Tools Biocytins for Neuron Staining
Standard Cat. No. Product Name Fluorophore Ex/Em (nm) filter set(s)
Alexa Fluor® NTA-2011-ZP87 Alexa Fluor® 488 Biocytin FITC 495/519 488
Alexa Fluor® NTA-2011-ZP88 Alexa Fluor® 546 Biocytin TRITC 556/573 546
Alexa Fluor® NTA-2011-ZP89 Alexa Fluor® 594 Biocytin Texas Red® 590/617 594
l Labeled Dextran Conjugates
Dextran is a natural synthetic polysaccharide with good water solubility, low toxicity, and different molecular weight. They have been successfully used as long-term tracers for living cells. Our dextran is purified by size chromatography to remove unbound molecules and ensure proper cell biology analysis applications. Dextran is functionalized with various groups, dyes, and other biomolecules.
Creative Biolabs provides a complete list of labeled dextran conjugates, which are available with a wide selection of colors and a range of molecular weights.
• Available conjugation: CF™488A; CF™543; CF™555; CF™568; CF™594; CF™640R; CF™680; CF™680R; CF™750; CF™770; CF™790
• Available WM: 3,000 WM; 5,000 WM; 10,000 WM; 40,000 WM; 70,000 WM; 500,000 WM; 2,000,000 WM
Virus Vector In Neural Circuit Tracing Page 40 03 Common Neural Circuit Tracing Tools Appendix
Dextran Conjugates, 3,000 MW
Standard Ex/Em Cat. No. Product Name Fluorophore filter set(s) (nm) Dextran, Biotin-Conjugated, NTA-2011-ZP90 NA Biotin NA 3,000 MW [Lysine-Fixable] Dextran, Fluorescein and NTA-2011-ZP91 Biotin-Conjugated, 3,000 FITC Fluorescein 494/518 MW [Lysine-Fixable] Dextran, Tetramethylrhodamine and NTA-2011-ZP92 TAMRA TAMRA 555/580 Biotin-Conjugated, 3,000 MW [Lysine-Fixable] Dextran, Fluorescein- NTA-2011-ZP93 Conjugated, 3,000 MW FITC Fluorescein 494/521 [Lysine-Fixable] Dextran, Tetramethylrhodamine- NTA-2011-ZP94 TAMRA TAMRA 555/580 Conjugated, 3,000 MW [Lysine-Fixable] Dextran, Texas Red®- Texas NTA-2011-ZP95 Conjugated, 3,000 MW Texas Red® 595/615 Red® [Lysine-Fixable] Dextran, Rhodamine Green- Rhodamine NTA-2011-ZP96 Conjugated, 3,000 MW FITC 502/527 Green [Non-Fixable] Dextran, Tetramethylrhodamine- NTA-2011-ZP97 TAMRA TAMRA 555/580 Conjugated, 3,000 MW [Non-Fixable] Dextran, Texas Red®- Texas NTA-2011-ZP98 Conjugated, 3,000 MW Texas Red® 595/615 Red® [Non-Fixable]
Virus Vector In Neural Circuit Tracing Page 41 03 Common Neural Circuit Tracing Tools Dextran Conjugates, 10,000 MW
Standard Fluorophor Ex/Em Cat. No. Product Name filter e (nm) set(s) Dextran, Alexa Fluor® Alexa NTA-2011-ZP99 488-Conjugated, 10,000 FITC 495/519 Fluor® 488 MW Dextran, Alexa Fluor® Alexa NTA-2011-ZP100 546-Conjugated, 10,000 556/573 Fluor® 546 MW TRITC Dextran, Alexa Fluor® Alexa NTA-2011-ZP101 555-Conjugated, 10,000 555/565 Fluor® 555 MW Dextran, Alexa Fluor® Rhodami Alexa NTA-2011-ZP102 568-Conjugated, 10,000 578/603 ne Fluor® 568 MW Dextran, Alexa Fluor® Texas Alexa NTA-2011-ZP103 594-Conjugated, 10,000 590/617 Red® Fluor® 594 MW Dextran, Alexa Fluor® Alexa NTA-2011-ZP104 647-Conjugated, 10,000 Cy®5 650/668 Fluor® 647 MW Dextran, Alexa Fluor® Alexa NTA-2011-ZP105 680-Conjugated, 10,000 Cy®5.5 679/702 Fluor® 680 MW Dextran, Tetramethylrhodamine NTA-2011-ZP106 TAMRA TAMRA 555/580 and Biotin-Conjugated, 10,000 MW Dextran, Fluorescein and NTA-2011-ZP107 Biotin-Conjugated, FITC Fluorescein 494/518 10,000 MW
Virus Vector In Neural Circuit Tracing Page 42 03 Common Neural Circuit Tracing Tools l Lipophilic Tracers
Lipophilic tracers are used to track neuronal projections in fixed tissue sections. The dye uniformly marks neurons through the lateral diffusion in the plasma membrane and is supplemented by the active dye transport process in living tissues. Lipophilic tracers can also be used for tissue sections, neurons in vivo, fixed tissue sections, and cultured cells. Use these lipophilic nerve tracers for anterograde and retrograde transport research.
Properties of Lipophilic Tracers
Tissue sections Yes
In vivo neurons Yes
Fixed tissue sections Yes
Cultured cells Yes
Cell loading Iontophoresis, microinjection, or crystal/paste placement
Staining mode Exogenous
Best for Cell populations
Transport Bidirectional
Synaptic transfer No
Gap junction transfer No
Virus Vector In Neural Circuit Tracing Page 43 03 Common Neural Circuit Tracing Tools Creative Biolabs provides lipophilic probes with unsaturated alkyl chains to speed up the diffusion process.
Popular Lipophilic Tracers
Standard Ex/Em Cat. No. Product Name Fluorophore Fixable Format filter set(s) (nm)
NTA-2011-ZP120 "DiI" - DiIC18(3), Solid Solid
NTA-2011-ZP121 "DiI" - DiIC18(3), Crystal Crystal
DiI 549/565 No NewTrace™ DiI Tissue- NTA-2011-ZP50 Labeling Paste - DiO, Paste DiI, DiD
DiI Cell-Labeling NTA-2011-ZP122 Rhodamine Solution Solution
NewTrace™ CM-DiI Dye, NTA-2011-ZP123 Solid Solid
NewTrace™ CM-DiI NTA-2011-ZP48 CM DiI 549/565 Yes Paste Tissue Labeling Paste
CM-DiI Cell-Labeling NTA-2011-ZP124 Solution Solution
NTA-2011-ZP125 "DiD" - DiIC18(5), Solid Cy®5 DiD 644/665 Solid
No
NTA-2011-ZP126 "DiR" - DiIC18(7), Solid Cy®5.5 DiR 750/780 Solid
Virus Vector In Neural Circuit Tracing Page 44 03 Common Neural Circuit Tracing Tools l Protein Tracers
Such neuronal stains include cholera toxin subunit B, wheat germ agglutinin (WGA), fluorescent conjugate of isolectin GS-IB4, and α- Bungarotoxin (BTX). Introduced by microinjection or iontophoresis, these probes are fixable and photostable retrograde tracers. They provide options for synaptic studies (WGA) and neuronal subtype (GS-IB4) differentiation.
Selection Guides for Protein Conjugates
WGA Conjugates
Standard Cat. No. Product Name Fluorophore Ex/Em (nm) filter set(s)
Wheat Germ Agglutinin (WGA), NTA-2011-ZP127 DAPI Alexa Fluor® 350 346/442 Alexa Fluor® 350-Conjugated
Wheat Germ Agglutinin (WGA), NTA-2011-ZP128 FITC Alexa Fluor® 488 495/519 Alexa Fluor® 488-Conjugated
Wheat Germ Agglutinin (WGA), NTA-2011-ZP129 TRITC Alexa Fluor® 555 555/565 Alexa Fluor® 555-Conjugated
Wheat Germ Agglutinin (WGA), NTA-2011-ZP130 Texas Red® Alexa Fluor® 594 590/617 Alexa Fluor® 594-Conjugated
Wheat Germ Agglutinin (WGA), NTA-2011-ZP131 Texas Red® Alexa Fluor® 633 632/647 Alexa Fluor® 633-Conjugated
Wheat Germ Agglutinin (WGA), NTA-2011-ZP132 Cy®5 Alexa Fluor® 647 650/668 Alexa Fluor® 647-Conjugated
Wheat Germ Agglutinin (WGA), NTA-2011-ZP133 Cy®5.5 Alexa Fluor® 680 679/702 Alexa Fluor® 680-Conjugated
Virus Vector In Neural Circuit Tracing Page 45 03 Common Neural Circuit Tracing Tools Cholera Toxin
Standard Cat. No. Product Name Fluorophore Ex/Em (nm) filter set(s)
NTA-2011-ZP134 Cholera Toxin Subunit B, Biotin-XX Biotin-XX NA NA Cholera Toxin Subunit B, Horseradish NTA-2011-ZP135 Horseradish Peroxidase peroxidase
Cholera Toxin Subunit B, Alexa Alexa Fluor® NTA-2011-ZP136 FITC 495/519 Fluor® 488 488
Cholera Toxin Subunit B, Alexa Alexa Fluor® NTA-2011-ZP137 TRITC 555/565 Fluor® 55 555
Cholera Toxin Subunit B, Alexa Alexa Fluor® NTA-2011-ZP138 Texas Red® 590/617 Fluor® 594 594
Cholera Toxin Subunit B, Alexa Alexa Fluor® NTA-2011-ZP139 Cy®5 650/668 Fluor® 647 647
Isolectin
Standard Cat. No. Product Name Fluorophore Ex/Em (nm) filter set(s)
NTA-2011-ZP140 Isolectin GS-IB4, biotin-XX NA Biotin-XX NA
Alexa Fluor® NTA-2011-ZP141 Isolectin GS-IB4 , Alexa Fluor® 488 FITC 495/519 488
Alexa Fluor® NTA-2011-ZP142 Isolectin GS-IB4 , Alexa Fluor® 568 Rhodamine 578/603 568
Alexa Fluor® NTA-2011-ZP143 Isolectin GS-IB4 , Alexa Fluor® 594 Texas Red® 590/617 594
Alexa Fluor® NTA-2011-ZP144 Isolectin GS-IB4 , Alexa Fluor® 647 Cy®5 650/668 647
Virus Vector In Neural Circuit Tracing Page 46 03 Common Neural Circuit Tracing Tools l Viral Tracers
Non-viral conventional tracers are effective tools for visualizing a large number of neural connections. However, the use of non-viral conventional tracers is mainly limited to mapping global connections. Most conventional tracers do not have sufficient resolution to reveal connectivity at the level of molecularly defined cell types. Instead, use genetic strategies to target viruses for specific cell types.
Creative Biolabs offers a variety of viral vectors to replace chemical tracers with targeted viral genetics.
Pseudorabies Virus(PRV)
Cat. No. Product Name Description
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-ZP14 PRV-CAG-EGFP Retrograde, multisynaptic
Virus Vector In Neural Circuit Tracing Page 47 03 Common Neural Circuit Tracing Tools Rabies Virus (RABV)
Cat. No. Product Name Description
Combined with AAV-TVA and AAV-RVG, NTA-2011-ZP1 RV-EnVA-△G-dsRed Retrograde, Monosynaptic
Combined with AAV-TVA and AAV-RVG, NTA-2011-ZP2 RV-EnVA-△G-GCaMP6s-dsRed Retrograde, Monosynaptic, Calcium signal detection
Combined with AAV-TVA and AAV-RVG, NTA-2011-ZP3 RV-EnVA-△G-mCherry Retrograde, Monosynaptic
Combined with AAV-TVA and AAV-RVG, NTA-2011-ZP4 RV-EnVA-△G-eGFP-synphRFP Retrograde, Monosynaptic
Combined with AAV-TVA and AAV-RVG, NTA-2011-ZP5 RV-EnVA-△G-ChR2-dsRed Retrograde, Monosynaptic
RV-EnVA-△G-pre-mGRASP- Combined with AAV-TVA and AAV-RVG, NTA-2011-ZP6 dsRed Retrograde, Monosynaptic
RV-EnVA-△G-post-mGRASP- Combined with AAV-TVA and AAV-RVG, NTA-2011-ZP7 dsRed Retrograde, Monosynaptic
Combined with AAV-RVG, Retrograde, NTA-2011-ZP8 RV-N2C(G)-△G-eGFP Monosynaptic
Combined with AAV-RVG, Retrograde, NTA-2011-ZP9 RV-N2C(G)-△G-dsRed Monosynaptic
RV-N2C(G)-△G-EGFP- Combined with AAV-RVG, Retrograde, NTA-2011-ZP10 synphRFP Monosynaptic
Virus Vector In Neural Circuit Tracing Page 48 03 Common Neural Circuit Tracing Tools Herpes Simplex Virus (HSV)
Cat. No. Product Name Description
NTA-2011-ZP15 HSV-EGFP Anterograde, multisynaptic
NTA-2011-ZP16 HSV-tdTomato Anterograde, multisynaptic
HSV-LSL-tdtomato-2a- NTA-2011-ZP17 Cre dependent, Anterograde, multisynaptic TK(H356) Combined with AAV-TK, anterograde NTA-2011-ZP18 HSV-ΔTK-hUbC-tdTomato monosynaptic Cre dependent, Combined with AAV-TK, NTA-2011-ZP19 HSV-ΔTK-LSL-tdTomato(H361) anterograde monosynaptic
Vesicular Stomatitis Virus (VSV)
Cat. No. Product Name Description
NTA-2011-ZP20 VSV-eGFP Anterograde, multisynaptic
NTA-2011-ZP21 VSV-mCherry Anterograde, multisynaptic
NTA-2011-ZP22 VSV-BFP Anterograde, multisynaptic
Combined with AAV-TVA and AAV-VSVG, NTA-2011-ZP23 VSV-EnVA-ΔG-eGFP anterograde monosynaptic Combined with AAV-TVA and AAV-VSVG, NTA-2011-ZP24 VSV-EnVA-ΔG-mCherry anterograde monosynaptic Combined with AAV-VSVG, anterograde NTA-2011-ZP25 VSV-ΔG-eGFP monosynaptic Combined with AAV-VSVG, anterograde NTA-2011-ZP26 VSV-ΔG-mCherry monosynaptic Combined with AAV-VSVG, anterograde NTA-2011-ZP27 VSV-ΔG-taueGFP monosynaptic Combined with AAV-VSVG, anterograde NTA-2011-ZP28 VSV-ΔG-taueGFP-ferritin monosynaptic
Please visit our website for more product information: https://neuros.creative-biolabs.com
Virus Vector In Neural Circuit Tracing Page 49 03 Common Neural Circuit Tracing Tools Contact Us
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