Technical Report | Published:

Functional imaging of hippocampal place cells at cellular resolution during virtual navigation

Nature Neuroscience volume 13, pages 14331440 (2010) | Download Citation

Abstract

Spatial navigation is often used as a behavioral task in studies of the neuronal circuits that underlie cognition, learning and memory in rodents. The combination of in vivo microscopy with genetically encoded indicators has provided an important new tool for studying neuronal circuits, but has been technically difficult to apply during navigation. Here we describe methods for imaging the activity of neurons in the CA1 region of the hippocampus with subcellular resolution in behaving mice. Neurons that expressed the genetically encoded calcium indicator GCaMP3 were imaged through a chronic hippocampal window. Head-restrained mice performed spatial behaviors in a setup combining a virtual reality system and a custom-built two-photon microscope. We optically identified populations of place cells and determined the correlation between the location of their place fields in the virtual environment and their anatomical location in the local circuit. The combination of virtual reality and high-resolution functional imaging should allow a new generation of studies to investigate neuronal circuit dynamics during behavior.

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References

  1. 1.

    & The hippocampus as a spatial map. Preliminary evidence from unit activity in the freely-moving rat. Brain Res. 34, 171–175 (1971).

  2. 2.

    & The hippocampus and memory for “what,” “where,” and “when.” Learn. Mem. 11, 397–405 (2004).

  3. 3.

    Hippocampal neurophysiology in the behaving animal. in The Hippocampus Book (ed. P. Andersen) 475–548 (Oxford University Press, Oxford, 2007).

  4. 4.

    , , & Place cells, spatial maps and the population code for memory. Curr. Opin. Neurobiol. 15, 738–746 (2005).

  5. 5.

    et al. Hippocampal cells encode places by forming small anatomical clusters. Neuroscience 166, 994–1007 (2010).

  6. 6.

    , , & The organization of spatial coding in the hippocampus: a study of neural ensemble activity. J. Neurosci. 9, 2764–2775 (1989).

  7. 7.

    , & Distribution of spatial and nonspatial information in dorsal hippocampus. Nature 402, 610–614 (1999).

  8. 8.

    et al. Independence of firing correlates of anatomically proximate hippocampal pyramidal cells. J. Neurosci. 21, RC134 (2001).

  9. 9.

    & Sub-millisecond firing synchrony of closely neighboring pyramidal neurons in hippocampal CA1 of rats during delayed non-matching to sample task. Front. Neural Circuits 3, 9 (2009).

  10. 10.

    , & Two-photon laser-scanning fluorescence microscopy. Science 248, 73–76 (1990).

  11. 11.

    , & Functional clustering of neurons in motor cortex determined by cellular resolution imaging in awake behaving mice. J. Neurosci. 29, 13751–13760 (2009).

  12. 12.

    , , , & Imaging large-scale neural activity with cellular resolution in awake, mobile mice. Neuron 56, 43–57 (2007).

  13. 13.

    et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat. Methods 6, 875–881 (2009).

  14. 14.

    , , & High-resolution in vivo imaging of hippocampal dendrites and spines. J. Neurosci. 24, 3147–3151 (2004).

  15. 15.

    , , & Intracellular dynamics of hippocampal place cells during virtual navigation. Nature 461, 941–946 (2009).

  16. 16.

    , , , & Rats are able to navigate in virtual environments. J. Exp. Biol. 208, 561–569 (2005).

  17. 17.

    & Photon upmanship: why multiphoton imaging is more than a gimmick. Neuron 18, 351–357 (1997).

  18. 18.

    , & Characterization of tissue-specific transcription by the human synapsin I gene promoter. Proc. Natl. Acad. Sci. USA 88, 3431–3435 (1991).

  19. 19.

    et al. Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system. Mol. Ther. 10, 302–317 (2004).

  20. 20.

    , & Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues. Nat. Methods 7, 141–147 (2010).

  21. 21.

    , & Automated analysis of cellular signals from large-scale calcium imaging data. Neuron 63, 747–760 (2009).

  22. 22.

    , , , & Independent component analysis of high-resolution imaging data identifies distinct functional domains. Neuroimage 34, 94–108 (2007).

  23. 23.

    , & Imaging input and output of neocortical networks in vivo. Proc. Natl. Acad. Sci. USA 102, 14063–14068 (2005).

  24. 24.

    , , & The functional microarchitecture of the mouse barrel cortex. PLoS Biol. 5, e189 (2007).

  25. 25.

    , & Population imaging of ongoing neuronal activity in the visual cortex of awake rats. Nat. Neurosci. 11, 749–751 (2008).

  26. 26.

    , & The contributions of position, direction, and velocity to single unit activity in the hippocampus of freely-moving rats. Exp. Brain Res. 52, 41–49 (1983).

  27. 27.

    et al. Hippocampal CA3 NMDA receptors are crucial for memory acquisition of one-time experience. Neuron 38, 305–315 (2003).

  28. 28.

    & Place cell discharge is extremely variable during individual passes of the rat through the firing field. Proc. Natl. Acad. Sci. USA 95, 3182–3187 (1998).

  29. 29.

    , & Trajectory encoding in the hippocampus and entorhinal cortex. Neuron 27, 169–178 (2000).

  30. 30.

    , & Spatial representations of hippocampal CA1 neurons are modulated by behavioral context in a hippocampus-dependent memory task. J. Neurosci. 27, 2416–2423 (2007).

  31. 31.

    , , & Hippocampal replay is not a simple function of experience. Neuron 65, 695–705 (2010).

  32. 32.

    , & Optical imaging of calcium accumulation in hippocampal pyramidal cells during synaptic activation. Nature 341, 533–536 (1989).

  33. 33.

    , , & Activity-dependent action potential invasion and calcium influx into hippocampal CA1 dendrites. Science 268, 297–300 (1995).

  34. 34.

    et al. Brain state– and cell type–specific firing of hippocampal interneurons in vivo. Nature 421, 844–848 (2003).

  35. 35.

    & Spatial selectivity and theta phase precession in CA1 interneurons. Hippocampus 17, 161–174 (2007).

  36. 36.

    & Discrete place fields of hippocampal formation interneurons. J. Neurophysiol. 97, 4152–4161 (2007).

  37. 37.

    Rats in Virtual Reality: The Development of an Advanced Method to Study Animal Behaviour (Eberhard-Karls-University, Tübingen, 2008).

  38. 38.

    Large-scale recording of neuronal ensembles. Nat. Neurosci. 7, 446–451 (2004).

  39. 39.

    & Dynamics of the hippocampal ensemble code for space. Science 261, 1055–1058 (1993).

  40. 40.

    et al. Intracellular features predicted by extracellular recordings in the hippocampus in vivo. J. Neurophysiol. 84, 390–400 (2000).

  41. 41.

    , & Genetic dissection of neural circuits. Neuron 57, 634–660 (2008).

  42. 42.

    Visualization of the spatial and temporal dynamics of intracellular signaling. Dev. Cell 4, 295–305 (2003).

  43. 43.

    & Experience-dependent structural synaptic plasticity in the mammalian brain. Nat. Rev. Neurosci. 10, 647–658 (2009).

  44. 44.

    & Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol. 2, e329 (2004).

  45. 45.

    , , , & Millisecond-timescale, genetically targeted optical control of neural activity. Nat. Neurosci. 8, 1263–1268 (2005).

  46. 46.

    , & Electrophysiology of hippocampal neurons. I. Sequential invasion and synaptic organization. J. Neurophysiol. 24, 225–242 (1961).

  47. 47.

    , , & A miniature head-mounted two-photon microscope. high-resolution brain imaging in freely moving animals. Neuron 31, 903–912 (2001).

  48. 48.

    et al. Visually evoked activity in cortical cells imaged in freely moving animals. Proc. Natl. Acad. Sci. USA 106, 19557–19562 (2009).

  49. 49.

    , & ScanImage: flexible software for operating laser scanning microscopes. Biomed. Eng. Online 2, 13 (2003).

  50. 50.

    , , & In vivo two-photon calcium imaging of neuronal networks. Proc. Natl. Acad. Sci. USA 100, 7319–7324 (2003).

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Acknowledgements

This work was supported by Princeton University, the National Institutes of Health (grant 5R01MH083686-03), The Howard Hughes Medical Institute, The Helen Hay Whitney Foundation and The Patterson Trust. We thank F. Collman for the brain motion correction algorithm.

Author information

Affiliations

  1. Department of Molecular Biology and Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, USA.

    • Daniel A Dombeck
    • , Christopher D Harvey
    •  & David W Tank
  2. Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia, USA.

    • Lin Tian
    •  & Loren L Looger

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Contributions

D.A.D. and D.W.T. designed the research. D.A.D. performed the imaging experiments and developed the chronic hippocampal window system and surgery/training sequences. D.W.T. designed and implemented the combined two-photon microscope and virtual reality instrumentation. D.A.D. and C.D.H. performed extracellular recording and optimized virtual reality training procedures. L.T. and L.L.L. provided AAV2/1-synapsin-1-GCaMP3. D.A.D. analyzed the data. D.A.D. and D.W.T. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Daniel A Dombeck or David W Tank.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–5

Videos

  1. 1.

    Supplementary Movie 1

    Z-series movie of the hippocampal region labeled with GCaMP3 virus infection. Movie begins in the external capsule/alveus (dense plexus of fibers) and then steps ventral in 5 micron increments through stratum oriens, stratum pyramidale and then ends in stratum radiatum (350 microns below the external capsule surface). The field is 200×100 microns.

  2. 2.

    Supplementary Movie 2

    Functional two-photon movie of a field of CA1 neurons in a mouse running back and forth along a virtual linear track. The two-photon time-series was acquired at 15fps and the movie is displayed at 30fps. The virtual linear track is shown at the bottom of the movie and a “^” indicates the position of the mouse along the linear track. This movie corresponds to the data shown in Fig. 2 (the 2 minute time period shown in Fig. 2b). The two-photon movie was made by coloring all of the pixels within a neuron's ROI a red intensity proportional to the value of the significant transient only trace corresponding to each frame (the red color was saturated at 35% changes). This red intensity was added to the still grey-scale image.

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DOI

https://doi.org/10.1038/nn.2648

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