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

Dombeck DA, Harvey CD, Tian L, Looger LL, Tank DW - Nat. Neurosci. (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, USA. ddombeck@princeton.edu

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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Imaging CA1 place cells in the dorsal hippocampus. a. Two-photon image of neuron cell bodies in stratum pyramidale of CA1 labeled with GCaMP3. The indicator is excluded from the nucleus13. ROIs for example cells are shown in red (right). b. GCaMP3 baseline subtracted ΔF/F traces are shown in black for a subset of the cells labeled in a (right). Red traces indicate significant calcium transients with <5% false positive error rates (see Methods). The position of the mouse along the virtual linear track and reward times are shown at the bottom. c. Expanded view of boxed region in (b). d. Mean ΔF/F versus linear track position for a subset of the cells labeled in a (right). e. A plot of mean ΔF/F versus linear track position for all of the cells labeled in a (right). f. Place cells are colored according to the location of their place fields along the virtual linear track. Only place cells with significant place fields during running in the positive direction are shown here.
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Figure 2: Imaging CA1 place cells in the dorsal hippocampus. a. Two-photon image of neuron cell bodies in stratum pyramidale of CA1 labeled with GCaMP3. The indicator is excluded from the nucleus13. ROIs for example cells are shown in red (right). b. GCaMP3 baseline subtracted ΔF/F traces are shown in black for a subset of the cells labeled in a (right). Red traces indicate significant calcium transients with <5% false positive error rates (see Methods). The position of the mouse along the virtual linear track and reward times are shown at the bottom. c. Expanded view of boxed region in (b). d. Mean ΔF/F versus linear track position for a subset of the cells labeled in a (right). e. A plot of mean ΔF/F versus linear track position for all of the cells labeled in a (right). f. Place cells are colored according to the location of their place fields along the virtual linear track. Only place cells with significant place fields during running in the positive direction are shown here.

Mentions: To optically identify and characterize place cells, we collected time-series movies (∼64ms/frame) of fields of view (∼200×100 microns) in the CA1 region of the hippocampus containing ∼80–100 neurons (Fig. 2a, left) while the mice navigated the virtual linear track. Time-series at a single location in CA1 were acquired for ∼9–13 minutes. In all, 47 time-series from ∼10 different labeled regions in 4 mice were analyzed. During the time-series acquisitions, rewards were acquired at a rate of 3.2±0.7 rewards/min with a mean distance run between rewards of 2.5±0.5 m, similar to reward rates during our previous electrophysiology experiments15. To extract the fluorescence versus time traces for the individual neurons, the movies were first corrected for brain motion that occurred during the acquisition using a 2-D cross correlation algorithm (see Methods). It was difficult to manually draw regions of interest (ROIs) around individual neurons because CA1 cell bodies are tightly packed together, GCaMP3 resides only in the cytoplasm (near the cell edges), and the axial extent of the imaging focal spot is likely >∼4 microns20. Instead, an automated cell identification method based on independent component analysis (ICA) and principal component analysis (PCA)21,22 was used. Regions of interest for 10 neurons identified using this procedure are shown in red for the example field of view shown in Fig. 2a (right). ΔF/F traces (Fig. 2b, black traces with red segments) were extracted from the ICA/PCA-defined ROIs. ΔF/F traces revealed a baseline periodically interrupted by calcium transients that varied in amplitude (mean peak ΔF/F=28±32%), consistent with a difference in the number of underlying action potentials13,23,24, and varied in duration (mean transient duration=1.2±1.1s), consistent with the summation of multiple transients12,13,25. Significant transients with <5% false positive error rates were identified11,12 and used in all subsequent analyses (see Methods) (Fig. 2b,c red traces). These traces were taken as a surrogate measure of spiking activity and are referred to as the temporal activity pattern of the neurons.


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

Dombeck DA, Harvey CD, Tian L, Looger LL, Tank DW - Nat. Neurosci. (2010)

Imaging CA1 place cells in the dorsal hippocampus. a. Two-photon image of neuron cell bodies in stratum pyramidale of CA1 labeled with GCaMP3. The indicator is excluded from the nucleus13. ROIs for example cells are shown in red (right). b. GCaMP3 baseline subtracted ΔF/F traces are shown in black for a subset of the cells labeled in a (right). Red traces indicate significant calcium transients with <5% false positive error rates (see Methods). The position of the mouse along the virtual linear track and reward times are shown at the bottom. c. Expanded view of boxed region in (b). d. Mean ΔF/F versus linear track position for a subset of the cells labeled in a (right). e. A plot of mean ΔF/F versus linear track position for all of the cells labeled in a (right). f. Place cells are colored according to the location of their place fields along the virtual linear track. Only place cells with significant place fields during running in the positive direction are shown here.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2967725&req=5

Figure 2: Imaging CA1 place cells in the dorsal hippocampus. a. Two-photon image of neuron cell bodies in stratum pyramidale of CA1 labeled with GCaMP3. The indicator is excluded from the nucleus13. ROIs for example cells are shown in red (right). b. GCaMP3 baseline subtracted ΔF/F traces are shown in black for a subset of the cells labeled in a (right). Red traces indicate significant calcium transients with <5% false positive error rates (see Methods). The position of the mouse along the virtual linear track and reward times are shown at the bottom. c. Expanded view of boxed region in (b). d. Mean ΔF/F versus linear track position for a subset of the cells labeled in a (right). e. A plot of mean ΔF/F versus linear track position for all of the cells labeled in a (right). f. Place cells are colored according to the location of their place fields along the virtual linear track. Only place cells with significant place fields during running in the positive direction are shown here.
Mentions: To optically identify and characterize place cells, we collected time-series movies (∼64ms/frame) of fields of view (∼200×100 microns) in the CA1 region of the hippocampus containing ∼80–100 neurons (Fig. 2a, left) while the mice navigated the virtual linear track. Time-series at a single location in CA1 were acquired for ∼9–13 minutes. In all, 47 time-series from ∼10 different labeled regions in 4 mice were analyzed. During the time-series acquisitions, rewards were acquired at a rate of 3.2±0.7 rewards/min with a mean distance run between rewards of 2.5±0.5 m, similar to reward rates during our previous electrophysiology experiments15. To extract the fluorescence versus time traces for the individual neurons, the movies were first corrected for brain motion that occurred during the acquisition using a 2-D cross correlation algorithm (see Methods). It was difficult to manually draw regions of interest (ROIs) around individual neurons because CA1 cell bodies are tightly packed together, GCaMP3 resides only in the cytoplasm (near the cell edges), and the axial extent of the imaging focal spot is likely >∼4 microns20. Instead, an automated cell identification method based on independent component analysis (ICA) and principal component analysis (PCA)21,22 was used. Regions of interest for 10 neurons identified using this procedure are shown in red for the example field of view shown in Fig. 2a (right). ΔF/F traces (Fig. 2b, black traces with red segments) were extracted from the ICA/PCA-defined ROIs. ΔF/F traces revealed a baseline periodically interrupted by calcium transients that varied in amplitude (mean peak ΔF/F=28±32%), consistent with a difference in the number of underlying action potentials13,23,24, and varied in duration (mean transient duration=1.2±1.1s), consistent with the summation of multiple transients12,13,25. Significant transients with <5% false positive error rates were identified11,12 and used in all subsequent analyses (see Methods) (Fig. 2b,c red traces). These traces were taken as a surrogate measure of spiking activity and are referred to as the temporal activity pattern of the neurons.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, USA. ddombeck@princeton.edu

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.

Show MeSH