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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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Labeling and imaging thalamocortical synapses on L4 or L5 barrel cortex neurons using array tomography and electron microscopy. (A1) Low power fluorescence photomicrograph of tdTomato-expressing TC axons and terminals in a fixed section of barrel cortex prepared from an 5–6 month old mouse in which AAV-tdTomato was stereotaxically injected in VPm 20–30 days earlier (inset left shows schematic of VPm injection). (A2) Low power fluorescence photomicrograph of GFP fluorescence in neurons in L4 barrel cortex in a fixed section from adult six3-CRE mouse in which AAV-FLEX-revGFP virus was injected into L4, 20-30 days earlier. (A3) Low power fluorescence photomicrograph of GFP fluorescence in L5 pyramidal neurons in fixed section from a 5–6 month old Thy1-YFPh mouse. (B) Schematic of LR white embedding and ultrathin serial sectioning procedure. (C) Schematic of immunostaining and light imaging of serial sections. (D1) Schematic of light and electron microscopic imaging on the same sections to assess accuracy of synapse detection and annotation with array tomography (performed on VPm-L4 only). (D2) Reconstructed L5 pyramidal neuron and annotated TC synapses from large-scale array tomography experiment (VPm-L5 only).
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Figure 1: Labeling and imaging thalamocortical synapses on L4 or L5 barrel cortex neurons using array tomography and electron microscopy. (A1) Low power fluorescence photomicrograph of tdTomato-expressing TC axons and terminals in a fixed section of barrel cortex prepared from an 5–6 month old mouse in which AAV-tdTomato was stereotaxically injected in VPm 20–30 days earlier (inset left shows schematic of VPm injection). (A2) Low power fluorescence photomicrograph of GFP fluorescence in neurons in L4 barrel cortex in a fixed section from adult six3-CRE mouse in which AAV-FLEX-revGFP virus was injected into L4, 20-30 days earlier. (A3) Low power fluorescence photomicrograph of GFP fluorescence in L5 pyramidal neurons in fixed section from a 5–6 month old Thy1-YFPh mouse. (B) Schematic of LR white embedding and ultrathin serial sectioning procedure. (C) Schematic of immunostaining and light imaging of serial sections. (D1) Schematic of light and electron microscopic imaging on the same sections to assess accuracy of synapse detection and annotation with array tomography (performed on VPm-L4 only). (D2) Reconstructed L5 pyramidal neuron and annotated TC synapses from large-scale array tomography experiment (VPm-L5 only).

Mentions: To study the subcellular distribution of TC synapses onto L5 pyramidal neurons in primary somatosensory barrel cortex with LSAT, we labeled pre- and post-synaptic groups of neurons with different fluorescent proteins (Figure 1A). Post-synaptic neurons were labeled in Thy1-YFP (type H) mice in which cortical L5 neurons sparsely express YFP (Feng et al., 2000). We labeled neurons in the ventral posteriomedial nucleus of thalamus (VPm), which project to barrel cortex, with adeno-associated virus (AAV) expressing tdTomato. Adult mice were stereotaxically injected with AAV. After ~4 weeks of expression the brain was fixed by transcardial perfusion. The primary somatosensory cortex was embedded in LR White resin. Serial ultrathin sections (200 nm) were made from the embedded block covering a volume of ~0.2 mm3. Each section was stained with an anti-synaptophysin antibody to label pre-synaptic terminals, and DsRed and GFP antibodies to enhance the signal from the encoded expressed pre- and post-synaptic fluorophores (Figures 1B,C). Sections were then imaged and reconstructed in three dimensions (Figure 1D2). In a separate experiment we quantified the accuracy and reliability of synapse identification by AT. For this work we focused on L4 because of the higher density of TC synapses. L4 neurons were labeled with GFP using an AAV expressing FLEX-reverse GFP (Atasoy et al., 2008) injected into primary somatosensory cortex of six3-CRE mice (which express CRE recombinase in L4, but not other neocortical layers, Liao and Xu, 2008). AT fluorescence microscopy and EM were performed on the same serial sections (Figure 1D1).


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)

Labeling and imaging thalamocortical synapses on L4 or L5 barrel cortex neurons using array tomography and electron microscopy. (A1) Low power fluorescence photomicrograph of tdTomato-expressing TC axons and terminals in a fixed section of barrel cortex prepared from an 5–6 month old mouse in which AAV-tdTomato was stereotaxically injected in VPm 20–30 days earlier (inset left shows schematic of VPm injection). (A2) Low power fluorescence photomicrograph of GFP fluorescence in neurons in L4 barrel cortex in a fixed section from adult six3-CRE mouse in which AAV-FLEX-revGFP virus was injected into L4, 20-30 days earlier. (A3) Low power fluorescence photomicrograph of GFP fluorescence in L5 pyramidal neurons in fixed section from a 5–6 month old Thy1-YFPh mouse. (B) Schematic of LR white embedding and ultrathin serial sectioning procedure. (C) Schematic of immunostaining and light imaging of serial sections. (D1) Schematic of light and electron microscopic imaging on the same sections to assess accuracy of synapse detection and annotation with array tomography (performed on VPm-L4 only). (D2) Reconstructed L5 pyramidal neuron and annotated TC synapses from large-scale array tomography experiment (VPm-L5 only).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: Labeling and imaging thalamocortical synapses on L4 or L5 barrel cortex neurons using array tomography and electron microscopy. (A1) Low power fluorescence photomicrograph of tdTomato-expressing TC axons and terminals in a fixed section of barrel cortex prepared from an 5–6 month old mouse in which AAV-tdTomato was stereotaxically injected in VPm 20–30 days earlier (inset left shows schematic of VPm injection). (A2) Low power fluorescence photomicrograph of GFP fluorescence in neurons in L4 barrel cortex in a fixed section from adult six3-CRE mouse in which AAV-FLEX-revGFP virus was injected into L4, 20-30 days earlier. (A3) Low power fluorescence photomicrograph of GFP fluorescence in L5 pyramidal neurons in fixed section from a 5–6 month old Thy1-YFPh mouse. (B) Schematic of LR white embedding and ultrathin serial sectioning procedure. (C) Schematic of immunostaining and light imaging of serial sections. (D1) Schematic of light and electron microscopic imaging on the same sections to assess accuracy of synapse detection and annotation with array tomography (performed on VPm-L4 only). (D2) Reconstructed L5 pyramidal neuron and annotated TC synapses from large-scale array tomography experiment (VPm-L5 only).
Mentions: To study the subcellular distribution of TC synapses onto L5 pyramidal neurons in primary somatosensory barrel cortex with LSAT, we labeled pre- and post-synaptic groups of neurons with different fluorescent proteins (Figure 1A). Post-synaptic neurons were labeled in Thy1-YFP (type H) mice in which cortical L5 neurons sparsely express YFP (Feng et al., 2000). We labeled neurons in the ventral posteriomedial nucleus of thalamus (VPm), which project to barrel cortex, with adeno-associated virus (AAV) expressing tdTomato. Adult mice were stereotaxically injected with AAV. After ~4 weeks of expression the brain was fixed by transcardial perfusion. The primary somatosensory cortex was embedded in LR White resin. Serial ultrathin sections (200 nm) were made from the embedded block covering a volume of ~0.2 mm3. Each section was stained with an anti-synaptophysin antibody to label pre-synaptic terminals, and DsRed and GFP antibodies to enhance the signal from the encoded expressed pre- and post-synaptic fluorophores (Figures 1B,C). Sections were then imaged and reconstructed in three dimensions (Figure 1D2). In a separate experiment we quantified the accuracy and reliability of synapse identification by AT. For this work we focused on L4 because of the higher density of TC synapses. L4 neurons were labeled with GFP using an AAV expressing FLEX-reverse GFP (Atasoy et al., 2008) injected into primary somatosensory cortex of six3-CRE mice (which express CRE recombinase in L4, but not other neocortical layers, Liao and Xu, 2008). AT fluorescence microscopy and EM were performed on the same serial sections (Figure 1D1).

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