Limits...
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.

Show MeSH
LSAT shows that TC synapses are concentrated on proximal basal dendrites of L5 pyramidal neurons. (A) Three dimensional Scholl analysis of dendritic structure of reconstructed L5 pyramidal neurons, and (B) Number of TC synapses as a function of path distance from center of soma. (C) Average density of TC synapses from all annotated neurons (aligned relative to cell bodies; false color scale) represented on top of all dendrites from all the traced neurons (white lines, superimposed; scale bar 100 μm). (D,E) Spatial distribution of the functional TC input mapped using sCRACM (Petreanu et al., 2009) (D), false color scale; reduced to 80% of size to compensate for the tissue shrinkage during LSAT processing) and distribution of TC synapses detected by LSAT (E) average from all annotated neurons aligned relative to pia; scale bar 100 μm). (F) Overlay of TC laminar distribution measured by sCRACM and LSAT.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3824245&req=5

Figure 8: LSAT shows that TC synapses are concentrated on proximal basal dendrites of L5 pyramidal neurons. (A) Three dimensional Scholl analysis of dendritic structure of reconstructed L5 pyramidal neurons, and (B) Number of TC synapses as a function of path distance from center of soma. (C) Average density of TC synapses from all annotated neurons (aligned relative to cell bodies; false color scale) represented on top of all dendrites from all the traced neurons (white lines, superimposed; scale bar 100 μm). (D,E) Spatial distribution of the functional TC input mapped using sCRACM (Petreanu et al., 2009) (D), false color scale; reduced to 80% of size to compensate for the tissue shrinkage during LSAT processing) and distribution of TC synapses detected by LSAT (E) average from all annotated neurons aligned relative to pia; scale bar 100 μm). (F) Overlay of TC laminar distribution measured by sCRACM and LSAT.

Mentions: Having established the accuracy of AT in detecting synapses, we next analyzed the distribution of the TC input to L5 pyramidal neurons in our LSAT data set. We first looked at the distribution of TC synapses relative to the soma and cortical layers. Although the dendritic trees of L5 pyramidal neurons transverse L5-L1, we found that most of the TC synapses onto this cell type are located within 200 μm of the soma (Figures 8A–C; 77.9 ± 9.4% of all TC synapses within a 200 μm path length from the soma, n = 8). We compared our high resolution anatomical map to a functional spatial distribution map of TC inputs onto the L5 pyramidal cells in barrel cortex from our previous study using the lower resolution channelrhodopsin-2-assisted circuit mapping approach (“sCRACM”) (Petreanu et al., 2009) (Figures 8D–F). The two methods reveal a similar concentration of TC input on the basal dendrites and proximal apical dendrites. However, sCRACM shows that the TC input strength on the apical dendrites in L4 and L3 is stronger than would be predicted from the anatomy. This difference suggests that synapse strength and/or dendritic properties are additional contributors to functional TC synaptic strength in L5 pyramids. For example, there is good evidence in cortical pyramidal neurons for synaptic strength changes normalizing input along dendritic trees and active dendritic properties regulating synaptic integration that could account for the anatomical and functional differences we observe (Magee, 2000).


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)

LSAT shows that TC synapses are concentrated on proximal basal dendrites of L5 pyramidal neurons. (A) Three dimensional Scholl analysis of dendritic structure of reconstructed L5 pyramidal neurons, and (B) Number of TC synapses as a function of path distance from center of soma. (C) Average density of TC synapses from all annotated neurons (aligned relative to cell bodies; false color scale) represented on top of all dendrites from all the traced neurons (white lines, superimposed; scale bar 100 μm). (D,E) Spatial distribution of the functional TC input mapped using sCRACM (Petreanu et al., 2009) (D), false color scale; reduced to 80% of size to compensate for the tissue shrinkage during LSAT processing) and distribution of TC synapses detected by LSAT (E) average from all annotated neurons aligned relative to pia; scale bar 100 μm). (F) Overlay of TC laminar distribution measured by sCRACM and LSAT.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: LSAT shows that TC synapses are concentrated on proximal basal dendrites of L5 pyramidal neurons. (A) Three dimensional Scholl analysis of dendritic structure of reconstructed L5 pyramidal neurons, and (B) Number of TC synapses as a function of path distance from center of soma. (C) Average density of TC synapses from all annotated neurons (aligned relative to cell bodies; false color scale) represented on top of all dendrites from all the traced neurons (white lines, superimposed; scale bar 100 μm). (D,E) Spatial distribution of the functional TC input mapped using sCRACM (Petreanu et al., 2009) (D), false color scale; reduced to 80% of size to compensate for the tissue shrinkage during LSAT processing) and distribution of TC synapses detected by LSAT (E) average from all annotated neurons aligned relative to pia; scale bar 100 μm). (F) Overlay of TC laminar distribution measured by sCRACM and LSAT.
Mentions: Having established the accuracy of AT in detecting synapses, we next analyzed the distribution of the TC input to L5 pyramidal neurons in our LSAT data set. We first looked at the distribution of TC synapses relative to the soma and cortical layers. Although the dendritic trees of L5 pyramidal neurons transverse L5-L1, we found that most of the TC synapses onto this cell type are located within 200 μm of the soma (Figures 8A–C; 77.9 ± 9.4% of all TC synapses within a 200 μm path length from the soma, n = 8). We compared our high resolution anatomical map to a functional spatial distribution map of TC inputs onto the L5 pyramidal cells in barrel cortex from our previous study using the lower resolution channelrhodopsin-2-assisted circuit mapping approach (“sCRACM”) (Petreanu et al., 2009) (Figures 8D–F). The two methods reveal a similar concentration of TC input on the basal dendrites and proximal apical dendrites. However, sCRACM shows that the TC input strength on the apical dendrites in L4 and L3 is stronger than would be predicted from the anatomy. This difference suggests that synapse strength and/or dendritic properties are additional contributors to functional TC synaptic strength in L5 pyramids. For example, there is good evidence in cortical pyramidal neurons for synaptic strength changes normalizing input along dendritic trees and active dendritic properties regulating synaptic integration that could account for the anatomical and functional differences we observe (Magee, 2000).

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