Direct Input and Output-viral vectors- Brain Case ()
Using viral vectors to study the inputs and outputs of neurons is the most important approach to elucidating the structure of neural circuits.
The brain's neural network is a complex structure composed of a large number of neurons with different shapes and characteristics connected through synapses. It is the structural basis for the brain's cognitive, emotional, memory, imagination and other activities. Mapping neuronal projections can track the flow of information between different brain areas.
Common methods for antrograde tracing
1. Anterograde tracer
Anterograde tracers are mainly taken up by neuronal somata and dendrites and then travel to the axon, thereby marking the area where the neuron projects. Classic anterograde tracers mainly include the following types:
1. Phytohemagglutinin (PHA-L) has the advantage of showing very detailed morphology of nerve fiber terminals and basically has no problem of passing fiber labeling.
2. Radioactively labeled amino acids. These amino acids are absorbed and incorporated into the proteins of neurons. By moving to the axon, they can also be released from the axon terminal and absorbed by the corresponding postsynaptic neuron.
Antrograde tracing is often used with in situ hybridization, immunostaining, or Nissl staining.
2. Anterograde
viral vectors
A limitation of classical anterograde viral vectors is that all cells at the injection site take up the tracer, so the projection pattern revealed by this method is an overall reflection of the projections from different types of neurons. As a neuron tracking reagent, viruses can be more rigorous in direction, can be combined with Cre-Lox technology to achieve cell type-specific labeling, and can also carry genetic tools (such as optogenetic tools, calcium-sensitive dyes, and gene editing tools, RNA interference tools, etc.) can play an important role in the study of neural circuit functions.
Some commonly used antrograde viral vectors that do not cross synapses include adeno-associated viruses of serotypes 2, 8, and 9, retroviruses, and lentiviruses packaged with VSV-G.
3. Case display
Example 1: Identification of brain-wide efferents from POMC neurons
Experimental animals : POMC-Cre mice
Viruses used : AAV9-DIO-mtdTomato, AAV9-DIO-EmGFP
Injection site and virus amount : ARC and NTS, volume 300nl each, titer 2.00E+12vg/mL
Detection method : In order to label axons better, perfusion was performed one month later and the samples were taken and imaged with fluorescence fiber microscope.
Figure 1. Identification of brain-wide efferents from POMC neurons (Minmin Luo, et al. Frontiers in Neuroanatomy. 2015)
Example 2: The role of Dyrk1a (dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A) in brain development
Laboratory animals : prenatal and postnatal mice
Plasmid used : lentiviral plasmid
Experimental method : Intrauterine electroporation was injected into the brain ventricle of E14.5 mice, and the mice were sacrificed at P0, and materials were collected and imaged.
Figure 2. Dyrk1a(WT) overexpression can delay the migration of neurons in the developing mouse embryonic cortex (ZLQiu, et al. Molecular Psychiatry.2018)
Common methods of retrograde tracing
1. Retrograde tracer
Retrograde tracers are usually taken up by axon terminals and then transported back to the cell body. Classic retrograde tracers include:
1. Horseradish peroxidase (HRP): used to track the fiber connections of peripheral nerves and central nervous system.
2. Cholera toxin subunit b (CTb): It is a very sensitive cis and reverse tracking agent. CTb is one of the subunits. It is a unit that binds to cell receptors. It is non-toxic and is more effective as a tracking agent.
Determining whether a tracer is being transported in the forward or reverse direction depends primarily on observation. Retrograde tracers usually bind to receptors that are selectively enriched in axonal terminals, are absorbed through endocytosis at the axonal terminals, and reach the cell body through the endogenous retrograde axonal transport system.
2. Retrograde viral vectors
Like the classic anterograde tracers, the above-mentioned traditional retrograde tracers are also directionally non-specific and cannot track specific types of neurons in the same target area.
Some commonly used retrograde viral vectors that do not cross synapses include
1. Adeno-associated virus AAV of serotype Retro has the advantages of high retrograde labeling efficiency, wide application, and great clinical application potential.
2. The recombinant rabies virus lacking the RV-G gene carries fluorescent protein genes such as GFP and mCherry, which allows foreign proteins to be expressed in high abundance in neurons, thereby clearly marking the fine morphology of neurons.
3. Canine adenovirus type 2 (CAV-2) has the advantages of low immunotoxicity and preferential infection of neurons. CAV-2 viruses are also helper virus-dependent virions with a clonal load of approximately 30 kb. These basic properties expand the role of CAV-2 in gene therapy of the central nervous system.
3. Case display
Example 1: Retrograde viral tracing of LC-SC projections
Experimental animals : WT mice
Detection method : AAV-Retro-Cre is injected in SC, AAV9-DIO-mCherry is injected in LC, and mCherry fluorescence signal can be detected in LC after 4 weeks.
Figure 3. Retrograde viral tracing of LC-SC projections (LipingWang, et al.Current Biology.2018)
Example 2: Separation of VTAergic neurons innervating NAC and mOT
Experimental animals : C57BL/6mice
Viruses used : RV-DG-DsRed and RV-DG-GFP, titer 10^8 TU
Experimental methods and results : 100nlRV-DG-DsRed and RV-DG-GFP were injected into NAC and mOT respectively, and after 1 week perfusion, neurons could be traced in the VTA.
Figure 4. Isolation of TA-ergic neurons innervating NAC and mOT (Zhijian Zhang, et al.eLife.2017)
Neural Circuit Tracing Services
Labeling Type
Synaptic Specificity
Common Tools
Retrograde Tracing
Non-synaptic
AAV2/Re, AAV2/11, RV-ΔG-N2cG, CTB
Retrograde Monosynaptic Tracing
Monosynaptic
RV-EnvA-ΔG-XFP
Retrograde Mutisynaptic Tracing
Mutisynaptic
PRV-XFP, PRV-△TK-DIO-XFP (Cre-dependent)
Anterograde Tracing
Non-synaptic
AAV2/2, AAV2/8, AAV2/9
Anterograde Monosynaptic Tracing
Monosynaptic
AAV2/1, AAV2/9-mWGA, Hs06, H361
Anterograde Mutisynaptic Tracing
Mutisynaptic
HSV, VSV
Deliverables (for all services above): Full project report and raw imaging data
If you are interested in the details of the experiment or the problems that may arise during the experiment and their causes, please contact: BD@ebraincase.com
