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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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Preparation conditions used for correlative electron microscopy preserve antigenicity while allowing reliable EM detection of synapses. (A–C) Comparison of number of synaptophysin-positive punctae using different fixation protocols. Representative light microscopic images of synaptophysin immunostaining in sections from brains fixed using the LSAT protocol (A) and the correlative EM protocol (B). Quantification of synaptophysin-positive punctae in the two conditions (C). (D–F) Comparison of the number of PSDs detected in EM images under the two fixation conditions. Representative electron micrographs from sections from brains fixed using a traditional EM fixation protocol (D) and the correlative EM fixation protocol (E). Red arrows show identified synapses. Quantifications of number of synapses detected PSD under traditional EM conditions and from four independently prepared samples using the correlative EM conditions (F).
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Figure 5: Preparation conditions used for correlative electron microscopy preserve antigenicity while allowing reliable EM detection of synapses. (A–C) Comparison of number of synaptophysin-positive punctae using different fixation protocols. Representative light microscopic images of synaptophysin immunostaining in sections from brains fixed using the LSAT protocol (A) and the correlative EM protocol (B). Quantification of synaptophysin-positive punctae in the two conditions (C). (D–F) Comparison of the number of PSDs detected in EM images under the two fixation conditions. Representative electron micrographs from sections from brains fixed using a traditional EM fixation protocol (D) and the correlative EM fixation protocol (E). Red arrows show identified synapses. Quantifications of number of synapses detected PSD under traditional EM conditions and from four independently prepared samples using the correlative EM conditions (F).

Mentions: A critical unresolved question concerns the accuracy and reliability of AT in assigning synapses to the correct dendrite because of the high density of synapses in the cortical neuropil (~1/μm3). Although theoretical work predicts that AT can detect synapses with a high degree of accuracy (Mishchenko, 2010), this has not been rigorously tested experimentally. Thus, it is currently unknown whether AT is a truly quantitative method for synapses detection in large volume reconstruction. To address this issue, we directly compared synapse detection by AT with detection using EM on the same sections. We performed this experiment on TC inputs to L4 neurons in barrel cortex because of the higher TC synapse density in this region. Traditional EM preparation techniques quench intrinsic fluorescence of GFP and greatly reduce antigenicity. Therefore we explored the parameter space for conditions that can sufficiently preserve intrinsic fluorescence and antigenicity to allow light imaging for AT while still retaining sufficient structure in EM to reliably detect synapses. We found that a low concentration of osmium (0.001%) provided enough structural preservation for transmission EM while preserving synaptophysin antigenicity and GFP fluorescence. We compared the detection of synaptophysin punctae in this “correlative” EM condition to L4 barrel cortex sections prepared using a more traditional EM protocol (Figure 5). There was no difference in the number of synaptophysin punctae between these conditions. We also compared the number of post-synaptic densities (PSDs) detected in the same sections using EM. Although the ability to image membranes was diminished under the correlative EM conditions, PSDs were reliably detected and we consistently found no difference in the number of PSDs detected in L4 barrel cortex compared to traditional EM processing. Therefore, our correlative EM conditions allowed us to directly quantify the number of synapses detected both by AT and EM on the same ultrathin sections.


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)

Preparation conditions used for correlative electron microscopy preserve antigenicity while allowing reliable EM detection of synapses. (A–C) Comparison of number of synaptophysin-positive punctae using different fixation protocols. Representative light microscopic images of synaptophysin immunostaining in sections from brains fixed using the LSAT protocol (A) and the correlative EM protocol (B). Quantification of synaptophysin-positive punctae in the two conditions (C). (D–F) Comparison of the number of PSDs detected in EM images under the two fixation conditions. Representative electron micrographs from sections from brains fixed using a traditional EM fixation protocol (D) and the correlative EM fixation protocol (E). Red arrows show identified synapses. Quantifications of number of synapses detected PSD under traditional EM conditions and from four independently prepared samples using the correlative EM conditions (F).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Preparation conditions used for correlative electron microscopy preserve antigenicity while allowing reliable EM detection of synapses. (A–C) Comparison of number of synaptophysin-positive punctae using different fixation protocols. Representative light microscopic images of synaptophysin immunostaining in sections from brains fixed using the LSAT protocol (A) and the correlative EM protocol (B). Quantification of synaptophysin-positive punctae in the two conditions (C). (D–F) Comparison of the number of PSDs detected in EM images under the two fixation conditions. Representative electron micrographs from sections from brains fixed using a traditional EM fixation protocol (D) and the correlative EM fixation protocol (E). Red arrows show identified synapses. Quantifications of number of synapses detected PSD under traditional EM conditions and from four independently prepared samples using the correlative EM conditions (F).
Mentions: A critical unresolved question concerns the accuracy and reliability of AT in assigning synapses to the correct dendrite because of the high density of synapses in the cortical neuropil (~1/μm3). Although theoretical work predicts that AT can detect synapses with a high degree of accuracy (Mishchenko, 2010), this has not been rigorously tested experimentally. Thus, it is currently unknown whether AT is a truly quantitative method for synapses detection in large volume reconstruction. To address this issue, we directly compared synapse detection by AT with detection using EM on the same sections. We performed this experiment on TC inputs to L4 neurons in barrel cortex because of the higher TC synapse density in this region. Traditional EM preparation techniques quench intrinsic fluorescence of GFP and greatly reduce antigenicity. Therefore we explored the parameter space for conditions that can sufficiently preserve intrinsic fluorescence and antigenicity to allow light imaging for AT while still retaining sufficient structure in EM to reliably detect synapses. We found that a low concentration of osmium (0.001%) provided enough structural preservation for transmission EM while preserving synaptophysin antigenicity and GFP fluorescence. We compared the detection of synaptophysin punctae in this “correlative” EM condition to L4 barrel cortex sections prepared using a more traditional EM protocol (Figure 5). There was no difference in the number of synaptophysin punctae between these conditions. We also compared the number of post-synaptic densities (PSDs) detected in the same sections using EM. Although the ability to image membranes was diminished under the correlative EM conditions, PSDs were reliably detected and we consistently found no difference in the number of PSDs detected in L4 barrel cortex compared to traditional EM processing. Therefore, our correlative EM conditions allowed us to directly quantify the number of synapses detected both by AT and EM on the same ultrathin sections.

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