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Thalamocortical input onto layer 5 pyramidal neurons measured using quantitative large-scale array tomography.

Rah JC, Bas E, Colonell J, Mishchenko Y, Karsh B, Fetter RD, Myers EW, Chklovskii DB, Svoboda K, Harris TD, Isaac JT - Front Neural Circuits (2013)

Bottom Line: We found that TC synapses primarily target basal dendrites in layer 5, but also make a considerable input to proximal apical dendrites in L4, consistent with previous work.Our analysis further suggests that TC inputs are biased toward certain branches and, within branches, synapses show significant clustering with an excess of TC synapse nearest neighbors within 5-15 μm compared to a random distribution.We anticipate that this technique will be of wide utility for mapping functionally-relevant anatomical connectivity in neural circuits.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Janelia Farm Research Campus Ashburn, VA, USA ; Developmental Synaptic Plasticity Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health Bethesda, MD, USA.

ABSTRACT
The subcellular locations of synapses on pyramidal neurons strongly influences dendritic integration and synaptic plasticity. Despite this, there is little quantitative data on spatial distributions of specific types of synaptic input. Here we use array tomography (AT), a high-resolution optical microscopy method, to examine thalamocortical (TC) input onto layer 5 pyramidal neurons. We first verified the ability of AT to identify synapses using parallel electron microscopic analysis of TC synapses in layer 4. We then use large-scale array tomography (LSAT) to measure TC synapse distribution on L5 pyramidal neurons in a 1.00 × 0.83 × 0.21 mm(3) volume of mouse somatosensory cortex. We found that TC synapses primarily target basal dendrites in layer 5, but also make a considerable input to proximal apical dendrites in L4, consistent with previous work. Our analysis further suggests that TC inputs are biased toward certain branches and, within branches, synapses show significant clustering with an excess of TC synapse nearest neighbors within 5-15 μm compared to a random distribution. Thus, we show that AT is a sensitive and quantitative method to map specific types of synaptic input on the dendrites of entire neurons. We anticipate that this technique will be of wide utility for mapping functionally-relevant anatomical connectivity in neural circuits.

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Simulation of accuracy of synapse detection by AT. (A) Simulated AT images using a previously segmented EM image data set [from Mishchenko et al. (2010)]. (A1) an example EM image; (A2) same image segmented into pre- and post-synaptic structures; (A3) segmented image was blurred to produce the same resolution as in the light microscopy of AT with 90% of structures removed to mimic the sparseness of fluorescent labeling of the AT images; (A4) The pixel size of A3 was adjusted to produce the same image as in the light microscopy in AT. Red indicates presynaptic structures, green postsynaptic, blue synaptophysin and red arrowheads indicates a synapse. Scale bar = 1 μm (B) Close-up of a simulated synapse showing postsynaptic structure (green) and simulated synaptophysin staining (blue). Note that the predicted synaptophysin staining exhibits an increasing intensity gradient toward the synaptic contact as found experimentally (Figure 10; Scale bar = 0.5 μm). (C) False positive rate in simulations of AT using different light imaging isotropic spatial resolutions. Black dashed line indicates resolution of the light imaging in the current study. Blue dotted line is an extrapolation of simulated data to infinite resolution.
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Figure 7: Simulation of accuracy of synapse detection by AT. (A) Simulated AT images using a previously segmented EM image data set [from Mishchenko et al. (2010)]. (A1) an example EM image; (A2) same image segmented into pre- and post-synaptic structures; (A3) segmented image was blurred to produce the same resolution as in the light microscopy of AT with 90% of structures removed to mimic the sparseness of fluorescent labeling of the AT images; (A4) The pixel size of A3 was adjusted to produce the same image as in the light microscopy in AT. Red indicates presynaptic structures, green postsynaptic, blue synaptophysin and red arrowheads indicates a synapse. Scale bar = 1 μm (B) Close-up of a simulated synapse showing postsynaptic structure (green) and simulated synaptophysin staining (blue). Note that the predicted synaptophysin staining exhibits an increasing intensity gradient toward the synaptic contact as found experimentally (Figure 10; Scale bar = 0.5 μm). (C) False positive rate in simulations of AT using different light imaging isotropic spatial resolutions. Black dashed line indicates resolution of the light imaging in the current study. Blue dotted line is an extrapolation of simulated data to infinite resolution.

Mentions: We next compared the experimentally-determined accuracy to that obtained from a simulation of our staining and imaging conditions. We first took a stack of EM images that had been segmented (Figure 7A1) (Mishchenko et al., 2010). The segmented volume was color coded as red (pre-synaptic structure), green (post-synaptic structure) or white (synaptic contact) (Figure 7A2). To simulate the corresponding fluorescence signal that would be generated by AT over a range of simulated light imaging resolutions out of the EM image, we did the following steps. The fluorescence signal was made sparse to simulate our experimental labeling, and synaptophysin staining of pre-synaptic vesicle clusters Figures 7A3,B1; blue) was added. We then blurred the image to recreate our experimental optical resolution (Figure 7A3) and finally increased the pixel size to that of our images (Figure 7A4). We then calculated the number of synaptophysin punctae observed to be apposed to each individual spine head at the different light imaging resolutions and compared the accuracy of this detection to the EM image to calculate a false positive rate for synapse detection by light microscopy (see Materials and Methods for further details). The modeling predicted ~30% false positive rate at our imaging resolution (200 nm) (Figure 7C), similar to the ~22% false positive rate observed experimentally.


Thalamocortical input onto layer 5 pyramidal neurons measured using quantitative large-scale array tomography.

Rah JC, Bas E, Colonell J, Mishchenko Y, Karsh B, Fetter RD, Myers EW, Chklovskii DB, Svoboda K, Harris TD, Isaac JT - Front Neural Circuits (2013)

Simulation of accuracy of synapse detection by AT. (A) Simulated AT images using a previously segmented EM image data set [from Mishchenko et al. (2010)]. (A1) an example EM image; (A2) same image segmented into pre- and post-synaptic structures; (A3) segmented image was blurred to produce the same resolution as in the light microscopy of AT with 90% of structures removed to mimic the sparseness of fluorescent labeling of the AT images; (A4) The pixel size of A3 was adjusted to produce the same image as in the light microscopy in AT. Red indicates presynaptic structures, green postsynaptic, blue synaptophysin and red arrowheads indicates a synapse. Scale bar = 1 μm (B) Close-up of a simulated synapse showing postsynaptic structure (green) and simulated synaptophysin staining (blue). Note that the predicted synaptophysin staining exhibits an increasing intensity gradient toward the synaptic contact as found experimentally (Figure 10; Scale bar = 0.5 μm). (C) False positive rate in simulations of AT using different light imaging isotropic spatial resolutions. Black dashed line indicates resolution of the light imaging in the current study. Blue dotted line is an extrapolation of simulated data to infinite resolution.
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Figure 7: Simulation of accuracy of synapse detection by AT. (A) Simulated AT images using a previously segmented EM image data set [from Mishchenko et al. (2010)]. (A1) an example EM image; (A2) same image segmented into pre- and post-synaptic structures; (A3) segmented image was blurred to produce the same resolution as in the light microscopy of AT with 90% of structures removed to mimic the sparseness of fluorescent labeling of the AT images; (A4) The pixel size of A3 was adjusted to produce the same image as in the light microscopy in AT. Red indicates presynaptic structures, green postsynaptic, blue synaptophysin and red arrowheads indicates a synapse. Scale bar = 1 μm (B) Close-up of a simulated synapse showing postsynaptic structure (green) and simulated synaptophysin staining (blue). Note that the predicted synaptophysin staining exhibits an increasing intensity gradient toward the synaptic contact as found experimentally (Figure 10; Scale bar = 0.5 μm). (C) False positive rate in simulations of AT using different light imaging isotropic spatial resolutions. Black dashed line indicates resolution of the light imaging in the current study. Blue dotted line is an extrapolation of simulated data to infinite resolution.
Mentions: We next compared the experimentally-determined accuracy to that obtained from a simulation of our staining and imaging conditions. We first took a stack of EM images that had been segmented (Figure 7A1) (Mishchenko et al., 2010). The segmented volume was color coded as red (pre-synaptic structure), green (post-synaptic structure) or white (synaptic contact) (Figure 7A2). To simulate the corresponding fluorescence signal that would be generated by AT over a range of simulated light imaging resolutions out of the EM image, we did the following steps. The fluorescence signal was made sparse to simulate our experimental labeling, and synaptophysin staining of pre-synaptic vesicle clusters Figures 7A3,B1; blue) was added. We then blurred the image to recreate our experimental optical resolution (Figure 7A3) and finally increased the pixel size to that of our images (Figure 7A4). We then calculated the number of synaptophysin punctae observed to be apposed to each individual spine head at the different light imaging resolutions and compared the accuracy of this detection to the EM image to calculate a false positive rate for synapse detection by light microscopy (see Materials and Methods for further details). The modeling predicted ~30% false positive rate at our imaging resolution (200 nm) (Figure 7C), similar to the ~22% false positive rate observed experimentally.

Bottom Line: We found that TC synapses primarily target basal dendrites in layer 5, but also make a considerable input to proximal apical dendrites in L4, consistent with previous work.Our analysis further suggests that TC inputs are biased toward certain branches and, within branches, synapses show significant clustering with an excess of TC synapse nearest neighbors within 5-15 μm compared to a random distribution.We anticipate that this technique will be of wide utility for mapping functionally-relevant anatomical connectivity in neural circuits.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Janelia Farm Research Campus Ashburn, VA, USA ; Developmental Synaptic Plasticity Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health Bethesda, MD, USA.

ABSTRACT
The subcellular locations of synapses on pyramidal neurons strongly influences dendritic integration and synaptic plasticity. Despite this, there is little quantitative data on spatial distributions of specific types of synaptic input. Here we use array tomography (AT), a high-resolution optical microscopy method, to examine thalamocortical (TC) input onto layer 5 pyramidal neurons. We first verified the ability of AT to identify synapses using parallel electron microscopic analysis of TC synapses in layer 4. We then use large-scale array tomography (LSAT) to measure TC synapse distribution on L5 pyramidal neurons in a 1.00 × 0.83 × 0.21 mm(3) volume of mouse somatosensory cortex. We found that TC synapses primarily target basal dendrites in layer 5, but also make a considerable input to proximal apical dendrites in L4, consistent with previous work. Our analysis further suggests that TC inputs are biased toward certain branches and, within branches, synapses show significant clustering with an excess of TC synapse nearest neighbors within 5-15 μm compared to a random distribution. Thus, we show that AT is a sensitive and quantitative method to map specific types of synaptic input on the dendrites of entire neurons. We anticipate that this technique will be of wide utility for mapping functionally-relevant anatomical connectivity in neural circuits.

Show MeSH
Related in: MedlinePlus