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Connection Input Mapping and 3D Reconstruction of the Brainst
AAV-EGFP (From BrainVTA ) was used for anterograde tracing to verify the retrograde tracing results. CB-555 (From BrainVTA) was used to label the CSF-contacting nucleus.
The viruses used in this article From BrainVTA are in the table below
Control  PT-0241 rAAV2/9-hSyn-EGFP
CB-555  Cholera toxin subunit B Alexa Fluor 555 conjugate
Pub Date: 2020-03-21, DOI: 10.3389/fncir.2020.00011 Email: sales@brainvta.com
Si-Yuan Song , Ying Li , Xiao-Meng Zhai , Yue-Hao Li , Cheng-Yi Bao , Cheng-Jing Shan, Jia Hong , Jun-Li Cao , Li-Cai Zhang
Objective: To investigate whether the CSF-contacting nucleus receives brainstem and spinal cord projections and to understand the functional significance of these connections.
Methods: The retrograde tracer cholera toxin B subunit (CB) was injected into the CSF-contacting nucleus in Sprague-Dawley rats according the previously reported stereotaxic coordinates. After 7–10 days, these rats were perfused and their brainstem and spinal cord were sliced (thickness, 40 mm) using a freezing microtome. All the sections were subjected to CB immunofluorescence staining. The distribution of CB-positive neuron in different brainstem and spinal cord areas was observed under fluorescence microscope.
Results: The retrograde labeled CB-positive neurons were found in the midbrain, pons, medulla oblongata, and spinal cord. Four functional areas including one hundred and twelve sub-regions have projections to the CSF-contacting nucleus. However, the density of CB-positive neuron distribution ranged from sparse to dense.
Conclusion: Based on the connectivity patterns of the CSF-contacting nucleus receives anatomical inputs from the brainstem and spinal cord, we preliminarily conclude and summarize that the CSF-contacting nucleus participates in pain, visceral activity, sleep and arousal, emotion, and drug addiction. The present study firstly illustrates the broad projections of the CSF-contacting nucleus from the brainstem and spinal cord, which implies the complicated functions of the nucleus especially for the unique roles of coordination in neural and body fluids regulation.

Fig.1 The injection of CB to demonstrate the functions and neural networks of the CSF-contacting nucleus 
To demonstrate the functions and neural networks of the CSF-contacting nucleus in the central nervous system, using immunofluorescence technique and anterograde tracer (From BrainVTA), the researchers preliminarily conclude and summarize that the CSF-contacting nucleus participates in pain, visceral activity,sleep and arousal, emotion, and drug addiction based on the connectivity patterns of the CSF-contacting nucleus receives anatomical inputs from the brainstem and spinal cord.

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