Two-photon calcium imaging of evoked activity from L5 somatosensory neurons in vivo


Multiphoton imaging (MPI) is widely used for recording activity simultaneously from many neurons in superficial cortical layers in vivo. We combined regenerative amplification multiphoton microscopy (RAMM) with genetically encoded calcium indicators to extend MPI of neuronal population activity into layer 5 (L5) of adult mouse somatosensory cortex. We found that this approach could be used to record and quantify spontaneous and sensory-evoked activity in populations of L5 neuronal somata located as much as 800 μm below the pia. In addition, we found that RAMM could be used to simultaneously image activity from large (80) populations of apical dendrites and follow these dendrites down to their somata of origin.

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Figure 1: Imaging L5 Somatosensory cortex neurons labeled with GCaMP3.
Figure 2: Sensory stimulation–evoked calcium transients from populations of L5 somata and dendrites in vivo.
Figure 3: L5 activity correlation structure.
Figure 4: Measuring activity in populations of dendrites.
Figure 5: Spontaneous activity in dendrites and its identification with the originating somata.


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We would like to thank P. Theer for technical help with the RegA, J. Sawinski for help with detection system fabrication and D.S. Greenberg for dendrite morphology analysis. This work was supported by the Max Planck Society.

Author information




W.M., D.J.W., W.D. and J.N.D.K. designed the research. W.M., D.J.W. and J.N.D.K. performed the research. D.J.W. and J.N.D.K. analyzed the data. U.C. performed the histological reconstructions. J.T.H., A.T.S. and L.L.L. produced the virus. W.M., D.J.W., J.N.D.K. and W.D. wrote the manuscript.

Corresponding author

Correspondence to Jason N D Kerr.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 3980 kb)

Supplementary Movie 1

Z-stack from pia to layer 5b (AVI 31015 kb)

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Mittmann, W., Wallace, D., Czubayko, U. et al. Two-photon calcium imaging of evoked activity from L5 somatosensory neurons in vivo. Nat Neurosci 14, 1089–1093 (2011).

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