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Original link:Metabiotics - Loop Tracing
The circuit connections between neurons are very complex, and a complete and accurate display of the structure and changes of these circuits is of great significance for revealing the causes of many diseases and developing new treatment methods.
Traditional neural tracing methods such as electron microscopy, Golgi staining, dyes, peptide markers, etc. can display the morphology and projection of neurons. A few tracers such as WGA, TTC, etc. can also achieve cross synaptic labeling. However, these methods have disadvantages such as indirect signal, non-specific direction, and severe signal attenuation after crossing synapses. Most importantly, these dyes cannot carry genes, so they can only be colored. Since Kristensson first applied herpes simplex virus (HSV) to label neurons across synapses in the 1980s, the application of various viral vectors in loop tracing has received increasing attention.
However, it should be noted that although these neurophilic viruses have strong infectivity and specificity for labeling neural circuits, their immunogenicity is strong, and animals often die a few days after injection, making behavioral testing or physiological research impossible. Currently, they are more suitable for searching for unknown neural circuits. In addition, such viruses require laboratories with a PII or higher level. Therefore, an increasing number of studies are using AAV for loop tracing labeling. Due to the lack of replication ability and extremely low immunogenicity of AAV, it does not cause significant toxicity or affect the normal physiology of animals. Therefore, AAV has become the most commonly used loop tracing tool as a viral vector.
Neurons are polarized cells, and information is transmitted from the axons of the higher-level neurons to the dendrites of the lower level neurons through synapses. Afterwards, information integration occurs between dendrites and cell bodies, and is emitted again through axons. Therefore, the labeling of neural circuits involves two fundamental issues: directionality (clockwise: labeling from cell body to axon; counterclockwise: labeling from axon to cell body) and whether it crosses synapses (Figure 1).
Figure 1: The target of neural loop tracing technology (modified from Peter L Strike), et al., eLS, 2011）
Application cases of loop tracing
1. Forward non transsynaptic labeling based on AAV
① Customers publish articles：Science. (IF=41.058). Mu D,et.al. (2017). A central neural circuit for itch sensation.[Adenovirus, itching, photogenetics, chemical genetics]
Injection site: PBN in mice
carrier：AAV-hSyn-HA-hM4Di-IRES-mCitrine
Serotype：AAV2/9
Virus titer:1.0× 1013 VG/mL
Injection volume: 150nl
Observation time: 3 weeks
② Customers publish articles：Neuron. (IF=14.319). Tian-Le Xu,et.al. (2019). Central Processing of Itch in the Midbrain Reward Center. [AAV, Itch, Photogenetics]
Injection site: Vgat Cre mouse VTA
carrier：AAV-EF1a-DIO-ChR2(H134R)-mCherry
Serotype：AAV2/8
Virus titer：7.39×1012 VG/mL
Injection volume:300-400nl
Observation time: 2-3 weeks
2. Reverse non synaptic labeling based on AAV - rAAV2 intro
① Customer published articles:Biological Psychiatry. (IF=11.984). Bing Xing Pan,et.al. (2018). Chronic stress causes projection-specific adaptation of amygdala neurons via SK channel downregulation. [Adenovirus, Anxiety Disorder, Photogenetics]
Injection site: mouse dmPFC, VHPC
Carrier:AAV2/1-retro-Syn-eYFP-Cre、AAV2/8-CaMKIIα-DO-SK2&AAV2/8-CaMKIIα-DIO-SK2-mCherry
Injection volume:300nl
Observation time: 4 weeks
② Internal test results
Injection site: Mouse BPN
carrier：rAAV2-retro-hSyn-EYFP
Observation time: 4 weeks
3. Cross synaptic labeling based on AAV
3.1 Forward Transsynaptic Labeling Based on AAV - rAAV2/1
① Customers publish articles：Nature Neuroscience. (IF=19.912). Yao J,et.al. (2018). A corticopontine circuit for initiation of urination. [Adenovirus, Neural Circuit]
Injection site: M1 and PMC in mouse cortex
Carrier:rAAV2/1-hSyn-Cre&rAAV2/9-DIO-hChR2(H134R)-mCherry、rAAV2/9-DIO-GCaMP6s
Virus titer:rAAV2/1: 5 × 1012 VG/mL；rAAV2/9-DIO-hChR2：1.2 × 1013 VG/mL；rAAV2/9-DIO-GCaMP6s：0.5 × 1012 VG/mL
Injection volume: Multi point injection, 30-40nl per point
Observation time: 4 weeks
② Customer published articles:Science Advances. (IF=11.511). Ping Zheng,et.al. (2019). Crucial role of feedback signals from prelimbic cortex to basolateral amygdala in the retrieval of morphine withdrawal memory. [腺相关病毒, 成瘾, AAV1, AAV-WGA-Cre]
Injection site: BLA in mouse cortex
carrier：rAAV2/1-hSyn-Cre-EGFP
Virus titer： 1.13 × 1013 VG/mL
Injection volume：60nl
Observation time: 5 weeks
3.2 Cross synaptic labeling using WGA—AAV-WGA-Cre
Customer published articles:Science Advances. (IF=11.511). Ping Zheng,et.al. (2019). Crucial role of feedback signals from prelimbic cortex to basolateral amygdala in the retrieval of morphine withdrawal memory.Adenovirus, addiction, AAV1, AAV-WGA-Cre]
Injection site: PrL in mice
Carrier:：AAV-hSyn-mCherry-IRES-WGA-Cre
Virus titer：4.28 × 1012 VG/mL
Injection volume：300nl
Observation time: 4 weeks
