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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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Combined electron and light microscopy to determine the accuracy of synapse detection by array tomography. (A–C) Transmission EM (A) and light microscopy (B) images from the same section of tissue, and the two images superimposed (C). Yellow contours in all three images show mitochondria that are used as fiducial markers for aligning images. Arrowheads indicate predicted TC synapses based on the light microscopic image, colors of arrowheads show true-false evaluation based on the EM image (as depicted in (D). (C1–C5) Close up images of TC synapses predicted by light microscopy. (D) Correlative light (D1–D3) and EM (D4–D6) images from the same serial sections. Red indicates thalamic axons, green postsynaptic structure, blue synaptophysin. Black dots in EM images are gold particles from immunogold staining for GFP. Red arrows indicate the PSD from the EM images used to identify. (E) Schematics of synapse assignments based on combined EM and light microscopy. (F) Quantitation of different synapse assignments based on combined EM and light microscopy (evaluation of 322 putative TC synapses). Scale bars = 1 μm.
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Figure 6: Combined electron and light microscopy to determine the accuracy of synapse detection by array tomography. (A–C) Transmission EM (A) and light microscopy (B) images from the same section of tissue, and the two images superimposed (C). Yellow contours in all three images show mitochondria that are used as fiducial markers for aligning images. Arrowheads indicate predicted TC synapses based on the light microscopic image, colors of arrowheads show true-false evaluation based on the EM image (as depicted in (D). (C1–C5) Close up images of TC synapses predicted by light microscopy. (D) Correlative light (D1–D3) and EM (D4–D6) images from the same serial sections. Red indicates thalamic axons, green postsynaptic structure, blue synaptophysin. Black dots in EM images are gold particles from immunogold staining for GFP. Red arrows indicate the PSD from the EM images used to identify. (E) Schematics of synapse assignments based on combined EM and light microscopy. (F) Quantitation of different synapse assignments based on combined EM and light microscopy (evaluation of 322 putative TC synapses). Scale bars = 1 μm.

Mentions: To directly compare synapse detection by AT and EM, we acquired light and electron microscopic images from the same stacks of serial sections (Figure 6). Since mitochondria are easily identified in EM and are GFP fluorescence-negative in light microscopy, we aligned the light and EM images using the mitochondria as unambiguous and abundant landmarks (Figures 6A–D). TC synapses in the stacks of images were then manually assigned using the light microscopy images. Once completed, the EM images of the same stacks of sections were then examined (blind to the light microscopy synapse assignment) and TC synapses assigned using this imaging modality. The combined light and EM image stacks were then examined together and TC synapses were classified into three categories using the EM image as ground truth (Figures 6C,E): (1) “True,” a pre-synaptic terminal containing red (tdTomato expressed in TC axon terminal) and blue (synaptophysin staining) apposed to a green spine (post-synaptic GFP expression) exhibiting a PSD; (2) “False positive,” pre-synaptic red and blue, post-synaptic green, but no detectable PSD apposed to pre-synaptic terminal; (note that these are sub-classified into two types based on whether a PSD is detected on the spine; Figure 6E), and (3) “False negative,” a TC pre-synaptic terminal (tdTomato and synaptophysin positive detected with light microscopy) apposed to a PSD detected by EM, but this synapse is not detected in light imaging. Using this approach, we analyzed 322 putative TC synapses from 4 independent experiments and found a false positive rate of 22 ± 8.0% and a false negative rate of 14.2 ± 3.1% (Figure 6F). Therefore under our conditions, using AT alone at least 78% of TC synapses are correctly identified, and 14% are not detected.


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)

Combined electron and light microscopy to determine the accuracy of synapse detection by array tomography. (A–C) Transmission EM (A) and light microscopy (B) images from the same section of tissue, and the two images superimposed (C). Yellow contours in all three images show mitochondria that are used as fiducial markers for aligning images. Arrowheads indicate predicted TC synapses based on the light microscopic image, colors of arrowheads show true-false evaluation based on the EM image (as depicted in (D). (C1–C5) Close up images of TC synapses predicted by light microscopy. (D) Correlative light (D1–D3) and EM (D4–D6) images from the same serial sections. Red indicates thalamic axons, green postsynaptic structure, blue synaptophysin. Black dots in EM images are gold particles from immunogold staining for GFP. Red arrows indicate the PSD from the EM images used to identify. (E) Schematics of synapse assignments based on combined EM and light microscopy. (F) Quantitation of different synapse assignments based on combined EM and light microscopy (evaluation of 322 putative TC synapses). Scale bars = 1 μm.
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Figure 6: Combined electron and light microscopy to determine the accuracy of synapse detection by array tomography. (A–C) Transmission EM (A) and light microscopy (B) images from the same section of tissue, and the two images superimposed (C). Yellow contours in all three images show mitochondria that are used as fiducial markers for aligning images. Arrowheads indicate predicted TC synapses based on the light microscopic image, colors of arrowheads show true-false evaluation based on the EM image (as depicted in (D). (C1–C5) Close up images of TC synapses predicted by light microscopy. (D) Correlative light (D1–D3) and EM (D4–D6) images from the same serial sections. Red indicates thalamic axons, green postsynaptic structure, blue synaptophysin. Black dots in EM images are gold particles from immunogold staining for GFP. Red arrows indicate the PSD from the EM images used to identify. (E) Schematics of synapse assignments based on combined EM and light microscopy. (F) Quantitation of different synapse assignments based on combined EM and light microscopy (evaluation of 322 putative TC synapses). Scale bars = 1 μm.
Mentions: To directly compare synapse detection by AT and EM, we acquired light and electron microscopic images from the same stacks of serial sections (Figure 6). Since mitochondria are easily identified in EM and are GFP fluorescence-negative in light microscopy, we aligned the light and EM images using the mitochondria as unambiguous and abundant landmarks (Figures 6A–D). TC synapses in the stacks of images were then manually assigned using the light microscopy images. Once completed, the EM images of the same stacks of sections were then examined (blind to the light microscopy synapse assignment) and TC synapses assigned using this imaging modality. The combined light and EM image stacks were then examined together and TC synapses were classified into three categories using the EM image as ground truth (Figures 6C,E): (1) “True,” a pre-synaptic terminal containing red (tdTomato expressed in TC axon terminal) and blue (synaptophysin staining) apposed to a green spine (post-synaptic GFP expression) exhibiting a PSD; (2) “False positive,” pre-synaptic red and blue, post-synaptic green, but no detectable PSD apposed to pre-synaptic terminal; (note that these are sub-classified into two types based on whether a PSD is detected on the spine; Figure 6E), and (3) “False negative,” a TC pre-synaptic terminal (tdTomato and synaptophysin positive detected with light microscopy) apposed to a PSD detected by EM, but this synapse is not detected in light imaging. Using this approach, we analyzed 322 putative TC synapses from 4 independent experiments and found a false positive rate of 22 ± 8.0% and a false negative rate of 14.2 ± 3.1% (Figure 6F). Therefore under our conditions, using AT alone at least 78% of TC synapses are correctly identified, and 14% are not detected.

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