Neural Circuit Tracing: Tools https://neuros.creative-biolabs.com DEDICATED 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