Advertisement

In Vivo Two-photon Calcium Imaging in Dendrites of Rabies Virus-labeled V1 Corticothalamic Neurons

  • Yajie Tang
  • Liang Li
  • Leqiang Sun
  • Jinsong Yu
  • Zhe Hu
  • Kaiqi Lian
  • Gang Cao
  • Jinxia DaiEmail author
METHOD
  • 153 Downloads

Abstract

Monitoring neuronal activity in vivo is critical to understanding the physiological or pathological functions of the brain. Two-photon Ca2+ imaging in vivo using a cranial window and specific neuronal labeling enables real-time, in situ, and long-term imaging of the living brain. Here, we constructed a recombinant rabies virus containing the Ca2+ indicator GCaMP6s along with the fluorescent protein DsRed2 as a baseline reference to ensure GCaMP6s signal reliability. This functional tracer was applied to retrogradely label specific V1–thalamus circuits and detect spontaneous Ca2+ activity in the dendrites of V1 corticothalamic neurons by in vivo two-photon Ca2+ imaging. Notably, we were able to record single-spine spontaneous Ca2+ activity in specific circuits. Distinct spontaneous Ca2+ dynamics in dendrites of V1 corticothalamic neurons were found for different V1–thalamus circuits. Our method can be applied to monitor Ca2+ dynamics in specific input circuits in vivo, and contribute to functional studies of defined neural circuits and the dissection of functional circuit connections.

Keywords

In vivo Ca2+ imaging Cranial window Two-photon microscopy Rabies virus Dendrite Primary visual cortex Corticothalamic projection Neural circuit tracing 

Notes

Acknowledgements

We thank Dr. Fuqiang Xu (Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences) and Dr. Edward Callaway (The SALK Institute, USA) for the rRV packing system. This work was supported by the National Natural Science Foundation of China (31700934 and 31371106).

Conflict of interest

The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Supplementary material

12264_2019_452_MOESM1_ESM.pdf (2.6 mb)
Supplementary material 1 (PDF 2708 kb)
12264_2019_452_MOESM2_ESM.avi (1 mb)
Supplementary material 2 (AVI 1047 kb)
12264_2019_452_MOESM3_ESM.avi (1.6 mb)
Supplementary material 3 (AVI 1611 kb)

References

  1. 1.
    Knoblich U, Huang L, Zeng H, Li L. Neuronal cell-subtype specificity of neural synchronization in mouse primary visual cortex. Nat Commun 2019, 10: 2533.CrossRefGoogle Scholar
  2. 2.
    Luo L, Callaway EM, Svoboda K. Genetic dissection of neural circuits. Neuron 2008, 57: 634–660.CrossRefGoogle Scholar
  3. 3.
    Huang L, Yuan T, Tan M, Xi Y, Hu Y, Tao Q, et al. A retinoraphe projection regulates serotonergic activity and looming-evoked defensive behaviour. Nat Commun 2017, 8: 14908.CrossRefGoogle Scholar
  4. 4.
    Zhang J, Tan L, Ren Y, Liang J, Lin R, Feng Q, et al. Presynaptic excitation via GABAB receptors in habenula cholinergic neurons regulates fear memory expression. Cell 2016, 166: 716–728.CrossRefGoogle Scholar
  5. 5.
    Yamada T, Yang Y, Valnegri P, Juric I, Abnousi A, Markwalter KH, et al. Sensory experience remodels genome architecture in neural circuit to drive motor learning. Nature 2019, 569: 708–713.CrossRefGoogle Scholar
  6. 6.
    Tomoda T, Sakurai T. Neural circuitry and brain functions. Clin Calcium 2018, 28: 844–850.PubMedGoogle Scholar
  7. 7.
    Shadmehr R. Distinct neural circuits for control of movement vs. holding still. J Neurophysiol 2017, 117: 1431–1460.CrossRefGoogle Scholar
  8. 8.
    Gobel W, Helmchen F. In vivo calcium imaging of neural network function. Physiology (Bethesda) 2007, 22: 358–365.Google Scholar
  9. 9.
    Yang W, Yuste R. In vivo imaging of neural activity. Nat Methods 2017, 14: 349–359.CrossRefGoogle Scholar
  10. 10.
    Tischbirek CH, Birkner A, Konnerth A. In vivo deep two-photon imaging of neural circuits with the fluorescent Ca(2+) indicator Cal-590. J Physiol 2017, 595: 3097–3105.CrossRefGoogle Scholar
  11. 11.
    Broussard GJ, Liang R, Tian L. Monitoring activity in neural circuits with genetically encoded indicators. Front Mol Neurosci 2014, 7: 97.CrossRefGoogle Scholar
  12. 12.
    Daigle TL, Madisen L, Hage TA, Valley MT, Knoblich U, Larsen RS, et al. A suite of transgenic driver and reporter mouse lines with enhanced brain-cell-type targeting and functionality. Cell 2018, 174: 465–480.e22.Google Scholar
  13. 13.
    Fosque BF, Sun Y, Dana H, Yang CT, Ohyama T, Tadross MR, et al. Labeling of active neural circuits in vivo with designed calcium integrators. Science 2015, 347: 755.CrossRefGoogle Scholar
  14. 14.
    Gazda K, Bazala M, Wegierski T. Microscopic imaging of calcium ions with genetically encoded calcium indicators. Postepy Biochem 2017, 63: 34–43.PubMedGoogle Scholar
  15. 15.
    Perry JL, Ramachandran NK, Utama B, Hyser JM. Use of genetically-encoded calcium indicators for live cell calcium imaging and localization in virus-infected cells. Methods 2015, 90: 28–38.CrossRefGoogle Scholar
  16. 16.
    Tada M, Takeuchi A, Hashizume M, Kitamura K, Kano M. A highly sensitive fluorescent indicator dye for calcium imaging of neural activity in vitro and in vivo. Eur J Neurosci 2014, 39: 1720–1728.CrossRefGoogle Scholar
  17. 17.
    Germond A, Fujita H, Ichimura T, Watanabe TM. Design and development of genetically encoded fluorescent sensors to monitor intracellular chemical and physical parameters. Biophys Rev 2016, 8: 121–138.CrossRefGoogle Scholar
  18. 18.
    Dana H, Chen TW, Hu A, Shields BC, Guo C, Looger LL, et al. Thy1-GCaMP6 transgenic mice for neuronal population imaging in vivo. PLoS One 2014, 9: e108697.CrossRefGoogle Scholar
  19. 19.
    Tian L, Hires SA, Looger LL. Imaging neuronal activity with genetically encoded calcium indicators. Cold Spring Harb Protoc 2012, 2012: pdb.top069609.Google Scholar
  20. 20.
    Dana H, Sun Y, Mohar B, Hulse BK, Kerlin AM, Hasseman JP, et al. High-performance calcium sensors for imaging activity in neuronal populations and microcompartments. Nat Methods 2019, 16: 649–657.CrossRefGoogle Scholar
  21. 21.
    Tervo DGR, Hwang BY, Viswanathan S, Gaj T, Lavzin M, Ritola KD, et al. A designer AAV variant permits efficient retrograde access to projection neurons. Neuron 2016, 92: 372–382.CrossRefGoogle Scholar
  22. 22.
    Chen SH, Haam J, Walker M, Scappini E, Naughton J, Martin NP. Production of viral constructs for neuroanatomy, calcium imaging, and optogenetics. Curr Protoc Neurosci 2019, 87: e66.CrossRefGoogle Scholar
  23. 23.
    Chen JL, Andermann ML, Keck T, Xu NL, Ziv Y. Imaging neuronal populations in behaving rodents: paradigms for studying neural circuits underlying behavior in the mammalian cortex. J Neurosci 2013, 33: 17631–17640.CrossRefGoogle Scholar
  24. 24.
    Peters AJ, Lee J, Hedrick NG, O’Neil K, Komiyama T. Reorganization of corticospinal output during motor learning. Nat Neurosci 2017, 20: 1133–1141.CrossRefGoogle Scholar
  25. 25.
    Ranganathan GN, Apostolides PF, Harnett MT, Xu NL, Druckmann S, Magee JC. Active dendritic integration and mixed neocortical network representations during an adaptive sensing behavior. Nat Neurosci 2018, 21: 1583–1590.CrossRefGoogle Scholar
  26. 26.
    Takahashi N, Oertner TG, Hegemann P, Larkum ME. Active cortical dendrites modulate perception. Science 2016, 354: 1587–1590.CrossRefGoogle Scholar
  27. 27.
    Yang G, Pan F, Parkhurst CN, Grutzendler J, Gan WB. Thinned-skull cranial window technique for long-term imaging of the cortex in live mice. Nat Protoc 2010, 5: 201–208.CrossRefGoogle Scholar
  28. 28.
    Holtmaat A, Bonhoeffer T, Chow DK, Chuckowree J, De Paola V, Hofer SB, et al. Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window. Nat Protoc 2009, 4: 1128–1144.CrossRefGoogle Scholar
  29. 29.
    Osakada F, Mori T, Cetin AH, Marshel JH, Virgen B, Callaway EM. New rabies virus variants for monitoring and manipulating activity and gene expression in defined neural circuits. Neuron 2011, 71: 617–631.CrossRefGoogle Scholar
  30. 30.
    Wilson DE, Whitney DE, Scholl B, Fitzpatrick D. Orientation selectivity and the functional clustering of synaptic inputs in primary visual cortex. Nat Neurosci 2016, 19: 1003–1009.CrossRefGoogle Scholar
  31. 31.
    Sofroniew NJ, Flickinger D, King J, Svoboda K. A large field of view two-photon mesoscope with subcellular resolution for in vivo imaging. Elife 2016, 5.Google Scholar
  32. 32.
    Birkner A, Tischbirek CH, Konnerth A. Improved deep two-photon calcium imaging in vivo. Cell Calcium 2017, 64: 29–35.CrossRefGoogle Scholar
  33. 33.
    Nemoto T. Development of novel two-photon microscopy for living brain and neuron. Microscopy (Oxf) 2014, 63 Suppl 1: i7–i8.CrossRefGoogle Scholar
  34. 34.
    Wickersham IR, Finke S, Conzelmann KK, Callaway EM. Retrograde neuronal tracing with a deletion-mutant rabies virus. Nat Methods 2007, 4: 47–49.CrossRefGoogle Scholar
  35. 35.
    Kim J, Matney CJ, Blankenship A, Hestrin S, Brown SP. Layer 6 corticothalamic neurons activate a cortical output layer, layer 5a. J Neurosci 2014, 34: 9656–9664.CrossRefGoogle Scholar
  36. 36.
    Svoboda K, Yasuda R. Principles of two-photon excitation microscopy and its applications to neuroscience. Neuron 2006, 50: 823–839.CrossRefGoogle Scholar
  37. 37.
    Wickersham IR, Lyon DC, Barnard RJO, Mori T, Finke S, Conzelmann KK, et al. Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons. Neuron 2007, 53: 639–647.CrossRefGoogle Scholar
  38. 38.
    Osakada F, Callaway EM. Design and generation of recombinant rabies virus vectors. Nat Protoc 2013, 8: 1583–1601.CrossRefGoogle Scholar
  39. 39.
    Ugolini G. Advances in viral transneuronal tracing. J Neurosci Methods 2010, 194: 2–20.CrossRefGoogle Scholar
  40. 40.
    Birkner A, Konnerth A. Deep two-photon imaging in vivo with a red-shifted calcium indicator. Methods Mol Biol 2019, 1929: 15–26.CrossRefGoogle Scholar
  41. 41.
    Zhang L, Liang B, Barbera G, Hawes S, Zhang Y, Stump K, et al. Miniscope GRIN lens system for calcium imaging of neuronal activity from deep brain structures in behaving animals. Cur Protoc Neurosci 2019, 86: e56.CrossRefGoogle Scholar

Copyright information

© Shanghai Institutes for Biological Sciences, CAS 2019

Authors and Affiliations

  • Yajie Tang
    • 1
    • 2
    • 3
  • Liang Li
    • 2
    • 3
  • Leqiang Sun
    • 2
    • 3
  • Jinsong Yu
    • 2
    • 3
  • Zhe Hu
    • 2
  • Kaiqi Lian
    • 1
  • Gang Cao
    • 2
    • 3
    • 4
    • 5
  • Jinxia Dai
    • 2
    • 3
    • 4
    • 5
    Email author
  1. 1.Academician Workstation of Animal Disease Control and Nutrition Immunity in Henan Province, Henan Joint International Research Laboratory of Veterinary Biologics Research and ApplicationAnyang Institute of TechnologyAnyangChina
  2. 2.State Key Laboratory of Agricultural MicrobiologyHuazhong Agricultural UniversityWuhanChina
  3. 3.College of Veterinary MedicineHuazhong Agricultural UniversityWuhanChina
  4. 4.Biomedical CenterHuazhong Agricultural UniversityWuhanChina
  5. 5.Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, College of Veterinary MedicineHuazhong Agricultural UniversityWuhanChina

Personalised recommendations