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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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TC synapse detection by AT. High power view of a section of dendrite of an YFP-expressing L5 pyramidal neuron with four example thalamo-recipient spines indicated (left panel; 3-D volume rendering of 13 serial sections = 2.6 μm thick). Right panels: images from three serial sections showing spine (YFP signal, green), TC terminal (tdTomato, red) and synaptophysin staining (blue) for each of the four indicated example TC synaptic contacts. Yellow arrowheads indicate locations of identified synaptic contacts (note that for synapses 2 and 4, the synaptic contact is detected in two of the images).
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Figure 3: TC synapse detection by AT. High power view of a section of dendrite of an YFP-expressing L5 pyramidal neuron with four example thalamo-recipient spines indicated (left panel; 3-D volume rendering of 13 serial sections = 2.6 μm thick). Right panels: images from three serial sections showing spine (YFP signal, green), TC terminal (tdTomato, red) and synaptophysin staining (blue) for each of the four indicated example TC synaptic contacts. Yellow arrowheads indicate locations of identified synaptic contacts (note that for synapses 2 and 4, the synaptic contact is detected in two of the images).

Mentions: For LSAT we prepared 1074 serial ribbon sections (thickness, 200 nm) of barrel cortex and immunostained each as above. Imaging was carried out on an automated Zeiss Observer inverted fluorescence microscope using a 100x, 1.45 N.A.objective and custom autofocussing (see Experimental procedures). Each section (area, 1.00 × 0.83 mm2) was imaged as a series of 240 overlapping tiles for a total of 257,760 images. The imaging time per tile was ~5.3 s and to image the entire volume required a total of ~877.5 h. The images were aligned in three dimensions using custom software and rendered in three-dimensions. The imaged volume contained the somata of ~56 labeled pyramidal neurons (Figures 2A–D; Supplementary movie 1). We chose eight neurons for manual reconstruction because large parts of their dendritic arbors were contained in the imaged volume (Figure 2E). In some neurons the apical tufts were severed. However, TC inputs from the VPm innervate L1 and L2 only sparsely (Jensen and Killackey, 1987; Zhang and Deschenes, 1998; Petreanu et al., 2009; Meyer et al., 2010; Oberlaender et al., 2012), suggesting that only a small fraction of TC input was missed. We also observed some enlargements of the TC axons, which may be result of overexpression of tdTomato. We identified putative TC synapses on these eight pyramidal neurons by manually inspecting the three-color image stacks. We scored YFP-labeled spines (green) touching tdTomato positive TC terminals (red) that were stained for synaptophysin (blue; Figure 3; see Materials and Methods). We reconstructed 4.7 ± 0.8 mm (mean ± standard deviation) dendrites and counted 328.0 ± 31.2 TC synapses per neuron (Figure 4).


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)

TC synapse detection by AT. High power view of a section of dendrite of an YFP-expressing L5 pyramidal neuron with four example thalamo-recipient spines indicated (left panel; 3-D volume rendering of 13 serial sections = 2.6 μm thick). Right panels: images from three serial sections showing spine (YFP signal, green), TC terminal (tdTomato, red) and synaptophysin staining (blue) for each of the four indicated example TC synaptic contacts. Yellow arrowheads indicate locations of identified synaptic contacts (note that for synapses 2 and 4, the synaptic contact is detected in two of the images).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3824245&req=5

Figure 3: TC synapse detection by AT. High power view of a section of dendrite of an YFP-expressing L5 pyramidal neuron with four example thalamo-recipient spines indicated (left panel; 3-D volume rendering of 13 serial sections = 2.6 μm thick). Right panels: images from three serial sections showing spine (YFP signal, green), TC terminal (tdTomato, red) and synaptophysin staining (blue) for each of the four indicated example TC synaptic contacts. Yellow arrowheads indicate locations of identified synaptic contacts (note that for synapses 2 and 4, the synaptic contact is detected in two of the images).
Mentions: For LSAT we prepared 1074 serial ribbon sections (thickness, 200 nm) of barrel cortex and immunostained each as above. Imaging was carried out on an automated Zeiss Observer inverted fluorescence microscope using a 100x, 1.45 N.A.objective and custom autofocussing (see Experimental procedures). Each section (area, 1.00 × 0.83 mm2) was imaged as a series of 240 overlapping tiles for a total of 257,760 images. The imaging time per tile was ~5.3 s and to image the entire volume required a total of ~877.5 h. The images were aligned in three dimensions using custom software and rendered in three-dimensions. The imaged volume contained the somata of ~56 labeled pyramidal neurons (Figures 2A–D; Supplementary movie 1). We chose eight neurons for manual reconstruction because large parts of their dendritic arbors were contained in the imaged volume (Figure 2E). In some neurons the apical tufts were severed. However, TC inputs from the VPm innervate L1 and L2 only sparsely (Jensen and Killackey, 1987; Zhang and Deschenes, 1998; Petreanu et al., 2009; Meyer et al., 2010; Oberlaender et al., 2012), suggesting that only a small fraction of TC input was missed. We also observed some enlargements of the TC axons, which may be result of overexpression of tdTomato. We identified putative TC synapses on these eight pyramidal neurons by manually inspecting the three-color image stacks. We scored YFP-labeled spines (green) touching tdTomato positive TC terminals (red) that were stained for synaptophysin (blue; Figure 3; see Materials and Methods). We reconstructed 4.7 ± 0.8 mm (mean ± standard deviation) dendrites and counted 328.0 ± 31.2 TC synapses per neuron (Figure 4).

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