Brain virus injection and optic fiber implantation for unraveling M1 layer-specific pathways for pain relief

Brain virus injection and optic fiber implantation for unraveling 

M1 layer-specific pathways for pain relief

Zheng Gan¹, Rohini Kuner¹

¹Pharmacology Institute, Medical Faculty Heidelberg, Heidelberg University, Heidelberg, Germany

Motor cortex stimulation (MCS) has been commonly applied in clinical for pain therapy. But the underlying neural circuits haven’t been thoroughly elucidated. The following protocols for brain injection and optic fiber implantation were employed in our study on revealing the layer-specific pathways of primary motor cortex (M1) for neuropathic pain alleviation via chemogenetics and optogenetics.

All the AAV virions employed were purchased from Addgene company and the viral vector facility of Zurich University, Switzerland. Details of the virions can be seen in Table 1. The title of each virion will be different from different batches, The non-viral tracer Fluorogold (CatLog number: 39286) was purchased from Sigma-Aldrich Chemie GmbH.  The coordinates for all the brain virus injections and fiber implantations were referred to the mouse atlas Paxinos and Franklin¹, and additional information on anatomical aspects and connectivity was derived from resources of the Allen Institute for Brain Science (ALLEN Mouse Brain Atlas – Version 2; available from: http://atlas.brain-map.org/). Details are provided in Table 2. Four injection positions and 2 depths/position were employed to target the whole hind-limb M1, while 4 positions with single injection were applied to target a specific M1 layer. Similarly, two injection positions were employed to thoroughly cover the ZI, rACC and the NAc areas each. Virus expression and the optical fiber position on each mouse was confirmed at the end of the experiments and mice with unsatisfactory virus expression and incorrect optical fiber implantation were excluded.

Tracing experiments with virions  

For anterograde tracing, AAV-CamKllα-mCherry or AAV-DIO-mCherry virions (40 nl/injection) were used on C57BL/6JRJ or Cux2, Rbp4 and Ntsr1-Cre mice to target the whole or layer specific hind-limb M1 area, respectively. For retrograde tracing, AAVrg-hSyn-YFP (1:1 dilution, 20 nl/injection), AAVrg-ChR2-YFP (20 nl/injection), AAVrg-ChR2-tdTomato virus (20 nl/injection) or the Fluorogold (CTB488, 50 nl/injection) was used on C57BL/6JRJ mice to address targeted brain domains. For trans-synaptic anterograde tracing, AAV1-EF1a-Flpo (30 nl/injection) and AAV1-EF1a-Coff/Fon-BFP (15 nl/injection) virions were used to target the hind-limb M1; AAV1-EF1a-fDIO-YFP (100 nl/injection) and AAV1-mDlx-mRuby² (100 nl/injection) virions to address the targeted post-synaptic domains^3,4.

Functional experiments

M1 and S1 functional experiments with DREADDs or optogenetics

For the M1 experiments with chemogenetics, AAV-Syn-hM3Dq-mCherry or AAV-CamKllα-hM3Dq-mCherry and the corresponding control AAV-Syn-mCherry or AAV-CamKllα-mCherry virions (40 nl/injection) were injected on C57BL/6JRJ mice to target the whole hind-limb M1. AAV-DIO-hM3Dq-mCherry virus (40 nl/injection) was injected on Cux2, Rbp4 and Ntsr1-Cre or PV and SOM-Cre mice to target specific layer or GABAergic subpopulation, respectively. For the M1 experiments with optogenetics, AAV-CamKllα-ChR2-YFP and the control AAV-CamKllα-YFP virions (40 nl/injection) was injected as well as two fibers were implanted (one was implanted vertically to target the anterior part of the area and the second one with 15° clockwise rotation) in C57BL/6JRJ mice to manipulate the whole M1. For the S1HL behaviour experiments with chemogenetics, AAV-Syn-hM3Dq-mCherry and the control AAV-Syn-mCherry (40 nl/injection) virions was employed on C57BL/6JRJ mice.

M1 pathways functional experiments with optogenetics

In experiments functionally addressing pathways from the M1 to ZI, PAG and MD_L, AAV-CamKllα-ChR2-YFP or AAV-DIO-ChR2-YFP (40 nl/injection) were injected in C57BL/6JRJ mice to target the whole M1 cortex or on Cux2, Rbp4 and Ntsr1-Cre mice to target M1 Layer 2/3, 5 and 6, while AAV-CamKllα-YFP or AAV-DIO-YFP (40 nl/injection) were used as control virions, respectively. For functional interrogation of the M1-MD-NAc, AAV1-hSyn-Cre (30 nl/injection) and AAV1-Con/Foff-BFP (15 nl/injection) virions were injected in the hind-limb M1, AAV-DIO-ChR2-YFP or AAV-DIO-YFP virions (100 nl) were injected in MD area to label the neurons in MD_L, which receive connection from M1 and then project to the NAc. Optical fibers (Doric Lenses; 200 μm, 0.39 NA, ThorLabs) were employed to target the selected areas for blue light stimulation to activate M1 efferent or MD_L efferent. All the optical fiber implants were inserted 0.3 mm above the targeted sites and fixed with the dental cement and a silver screw. For thoroughly addressing ZI as well as NAcC, two fibers were also applied: one was implanted vertically to target the anterior part of the area; the second one with 15° nose-down rotation to target the posterior part of ZI and with 15° anti-clockwise rotation to target the posterior part of NAcC, respectively. All the mice were kept at least 3 weeks after the brain injection for thorough virus expression.

Fiber photometry of MD-NAc connection

For the MD_L to NAc pathway fiber photometry, the mice were stereotatically injected with AAV-Syn-ChrimsonR-tdTomato virus (50 nl) in MD_L and pGP-AAV-syn-jGCaMP7f-WPRE virus (150 nl) in NAcC^5,6. An optical recording fiber (400 μm, 0.6 NA) and a stimulation optic fiber (Doric Lenses; 200 μm, 0.39 NA, ThorLabs) were implanted immediately after brain injection and cemented together with a custom-made head-bar using Superbond Cement System (C&B, Sun Medical Co. Ltd., Japan). For slice electrophysiology recordings in M1 pathways, C57BL/6JRJ mice were injected with AAV-CamKllα-hM3Dq-mCherry or AAV-CamKllα-ChR2-YFP virions (40 nl/injection) in hind-limb M1, respectively. For the MD_L-NAcC pathway slice electrophysiology recordings, GAD-Cre mice were injected with AAV-CamKllα-ChR2-YFP virus (50 nl) in the MD_L and AAV-DIO-mCherry virus (150 nl/injection) in NAcC to label the MSN neurons. 

Acknowledgements

The study was funded by the German Research Foundation grant in Collaborative Research Center 1158. The authors are grateful to Nadine Gehrig, Dunja Baumgartl-Ahlert, and Lirong Wang for technical assistance. The authors gratefully acknowledge the Interdisciplinary Neurobehavioral Core Facility of Medical Faculty Heidelberg for assistance with behavioral experiments.

Competing interests

The authors declare no competing interests

References

1.         G. Paxinos, K. B. J. Franklin, The mouse brain in stereotaxic coordinates.  (London, Academic Press, ed. 2nd, 2001).

2.         J. Dimidschstein et al., Nature Neuroscience 19, 1743 (2016).

3.         B. Zingg et al., Neuron 93, 33 (2017).

4.         B. Zingg, B. Peng, J. Huang, H. W. Tao, L. I. Zhang, Journal of neuroscience 40, 3250 (2020).

5.         H. Dana et al., Nature Methods 16, 649 (2019).

6.         N. C. Klapoetke et al., Nature Methods 11, 338 (2014).

Table 1. Information on all virions employed in this study

Table 2. Information on coordinates employed for analyzing and targeting specific anatomical divisions of the brain