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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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Related in: MedlinePlus

Clustering of TC input on dendrites of L5 pyramidal neurons. (A) Example of dendritic branches of the same L5 pyramidal cell (from dotted box in Figure 4B) showing different densities of TC synapses. (B) Examples of basal dendrites from simulated random synapses and the experimentally observed TC synapse distribution for the same neuron. The location of synapses is indicated by the red stars, synapse density for each branch is color-coded; view is from above (scale bar = 100 μm). (C) The relationships between length of dendritic branch and number of TC synapses onto L5 basal dendrites. Shaded areas with dashed lines denote 95% confidence intervals from simulated randomly distributed synapses of L5 basal dendrites. (D) Histogram of TC synapse density for all dendritic branches from all reconstructed neurons. Insets: expansions of peak and tail of the distribution as indicated by the dashed boxes. (E) Averaged cumulative distributions for nearest neighbor distances of TC synapses from L5 basal dendrites, for experimental data (red, eight reconstructed neurons) and for simulations of 8000 model neurons with randomly distributed synapses (black). (F) The mean clustering coefficient for TC synapses at various spatial thresholds from basal dendrites of all eight reconstructed neurons (red line) and for simulated randomly distributed synapses (black line). Bars (right y-axis) show statistical results using a KS test to determine the fraction of simulations there are significantly different from the experimental data set of whole neurons (open bars) or of L5 basal dendrites.
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Figure 9: Clustering of TC input on dendrites of L5 pyramidal neurons. (A) Example of dendritic branches of the same L5 pyramidal cell (from dotted box in Figure 4B) showing different densities of TC synapses. (B) Examples of basal dendrites from simulated random synapses and the experimentally observed TC synapse distribution for the same neuron. The location of synapses is indicated by the red stars, synapse density for each branch is color-coded; view is from above (scale bar = 100 μm). (C) The relationships between length of dendritic branch and number of TC synapses onto L5 basal dendrites. Shaded areas with dashed lines denote 95% confidence intervals from simulated randomly distributed synapses of L5 basal dendrites. (D) Histogram of TC synapse density for all dendritic branches from all reconstructed neurons. Insets: expansions of peak and tail of the distribution as indicated by the dashed boxes. (E) Averaged cumulative distributions for nearest neighbor distances of TC synapses from L5 basal dendrites, for experimental data (red, eight reconstructed neurons) and for simulations of 8000 model neurons with randomly distributed synapses (black). (F) The mean clustering coefficient for TC synapses at various spatial thresholds from basal dendrites of all eight reconstructed neurons (red line) and for simulated randomly distributed synapses (black line). Bars (right y-axis) show statistical results using a KS test to determine the fraction of simulations there are significantly different from the experimental data set of whole neurons (open bars) or of L5 basal dendrites.

Mentions: We next evaluated whether there was preferential targeting of certain dendritic branches by TC input. Such clustering of input on specific dendritic branches has important implications for integration of synaptic input and synaptic plasticity (Wei, 2001; Ariav et al., 2003; Polsky et al., 2004; Losonczy and Magee, 2006), but has been difficult to evaluate because of a lack of available techniques. At a coarse level it was noticeable that L5 cells do not receive TC input uniformly, with some branches receiving denser input than others (see Figure 4). To study the uniformity of TC input we compared the distribution of TC synapses on each neuron to simulated random distributions for the same neurons. One potential confound in this analysis, however, is differences in laminar distributions of TC synapses onto L5 pyramidal neurons. TC inputs from VPm onto L5 pyramidal cells occur primarily within L4, L5B, and L6 (Bernardo and Woolsey, 1987; Bureau et al., 2006; Oberlaender et al., 2012) and Figure 4. Therefore, it is possible that any apparent preference of TC synapses toward a subset of dendrites (compared to a random distribution across the whole dendritic tree) could be due to this layer-specific distribution rather than being specific to the TC input per se. Therefore, to control for this, we compared TC input to simulated random input onto basal dendrites only, which reside primarily within L5 (Figure 9B). If all branches have an equal probability of receiving TC input then there will be a very close relationship between dendritic branch length and TC synapse number. If not then “TC preferring” or “non-preferring” branches (e.g., blue vs. red branches in Figure 9A) will generate scatter away from the line of unity in a branch length vs. TC synapse number plot. To quantify this, we plotted the number of synapses vs. branch length from the eight reconstructed neurons (Figure 9C, closed circles) and overlaid that with the confidence interval determined from a simulated random distribution (Figure 9C, dotted lines and shaded area, 95% confidence level). 15.0% of dendritic branches were found to have a synaptic density outside the 95% confidence interval of the random distribution. This analysis shows that the experimental data set contains branches with an excess of TC synapses (above the shaded area) and a fraction with a lack of TC synapses (below shaded area) compared to the random distribution. We also evaluated preferential dendrite targeting by the TC input by plotting the normalized histogram for TC synapse density for individual dendritic branches, comparing the simulated data set for randomly distributed synapses with the experimental data set. This analysis showed that compared with the randomly distributed synapses, the experimental data exhibited a larger fraction of branches at the extremes of the distribution, i.e., that have no TC synapses or a high TC synapse density (Figure 9D, Kolmogorov-Smirnov (KS) test, significant, p = 1.1 × 10−37). These findings therefore indicate that TC afferents do not have equal preference for all basal dendritic branches on L5 pyramidal neurons but, instead, are biased toward a subset of preferred dendritic branches.


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)

Clustering of TC input on dendrites of L5 pyramidal neurons. (A) Example of dendritic branches of the same L5 pyramidal cell (from dotted box in Figure 4B) showing different densities of TC synapses. (B) Examples of basal dendrites from simulated random synapses and the experimentally observed TC synapse distribution for the same neuron. The location of synapses is indicated by the red stars, synapse density for each branch is color-coded; view is from above (scale bar = 100 μm). (C) The relationships between length of dendritic branch and number of TC synapses onto L5 basal dendrites. Shaded areas with dashed lines denote 95% confidence intervals from simulated randomly distributed synapses of L5 basal dendrites. (D) Histogram of TC synapse density for all dendritic branches from all reconstructed neurons. Insets: expansions of peak and tail of the distribution as indicated by the dashed boxes. (E) Averaged cumulative distributions for nearest neighbor distances of TC synapses from L5 basal dendrites, for experimental data (red, eight reconstructed neurons) and for simulations of 8000 model neurons with randomly distributed synapses (black). (F) The mean clustering coefficient for TC synapses at various spatial thresholds from basal dendrites of all eight reconstructed neurons (red line) and for simulated randomly distributed synapses (black line). Bars (right y-axis) show statistical results using a KS test to determine the fraction of simulations there are significantly different from the experimental data set of whole neurons (open bars) or of L5 basal dendrites.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 9: Clustering of TC input on dendrites of L5 pyramidal neurons. (A) Example of dendritic branches of the same L5 pyramidal cell (from dotted box in Figure 4B) showing different densities of TC synapses. (B) Examples of basal dendrites from simulated random synapses and the experimentally observed TC synapse distribution for the same neuron. The location of synapses is indicated by the red stars, synapse density for each branch is color-coded; view is from above (scale bar = 100 μm). (C) The relationships between length of dendritic branch and number of TC synapses onto L5 basal dendrites. Shaded areas with dashed lines denote 95% confidence intervals from simulated randomly distributed synapses of L5 basal dendrites. (D) Histogram of TC synapse density for all dendritic branches from all reconstructed neurons. Insets: expansions of peak and tail of the distribution as indicated by the dashed boxes. (E) Averaged cumulative distributions for nearest neighbor distances of TC synapses from L5 basal dendrites, for experimental data (red, eight reconstructed neurons) and for simulations of 8000 model neurons with randomly distributed synapses (black). (F) The mean clustering coefficient for TC synapses at various spatial thresholds from basal dendrites of all eight reconstructed neurons (red line) and for simulated randomly distributed synapses (black line). Bars (right y-axis) show statistical results using a KS test to determine the fraction of simulations there are significantly different from the experimental data set of whole neurons (open bars) or of L5 basal dendrites.
Mentions: We next evaluated whether there was preferential targeting of certain dendritic branches by TC input. Such clustering of input on specific dendritic branches has important implications for integration of synaptic input and synaptic plasticity (Wei, 2001; Ariav et al., 2003; Polsky et al., 2004; Losonczy and Magee, 2006), but has been difficult to evaluate because of a lack of available techniques. At a coarse level it was noticeable that L5 cells do not receive TC input uniformly, with some branches receiving denser input than others (see Figure 4). To study the uniformity of TC input we compared the distribution of TC synapses on each neuron to simulated random distributions for the same neurons. One potential confound in this analysis, however, is differences in laminar distributions of TC synapses onto L5 pyramidal neurons. TC inputs from VPm onto L5 pyramidal cells occur primarily within L4, L5B, and L6 (Bernardo and Woolsey, 1987; Bureau et al., 2006; Oberlaender et al., 2012) and Figure 4. Therefore, it is possible that any apparent preference of TC synapses toward a subset of dendrites (compared to a random distribution across the whole dendritic tree) could be due to this layer-specific distribution rather than being specific to the TC input per se. Therefore, to control for this, we compared TC input to simulated random input onto basal dendrites only, which reside primarily within L5 (Figure 9B). If all branches have an equal probability of receiving TC input then there will be a very close relationship between dendritic branch length and TC synapse number. If not then “TC preferring” or “non-preferring” branches (e.g., blue vs. red branches in Figure 9A) will generate scatter away from the line of unity in a branch length vs. TC synapse number plot. To quantify this, we plotted the number of synapses vs. branch length from the eight reconstructed neurons (Figure 9C, closed circles) and overlaid that with the confidence interval determined from a simulated random distribution (Figure 9C, dotted lines and shaded area, 95% confidence level). 15.0% of dendritic branches were found to have a synaptic density outside the 95% confidence interval of the random distribution. This analysis shows that the experimental data set contains branches with an excess of TC synapses (above the shaded area) and a fraction with a lack of TC synapses (below shaded area) compared to the random distribution. We also evaluated preferential dendrite targeting by the TC input by plotting the normalized histogram for TC synapse density for individual dendritic branches, comparing the simulated data set for randomly distributed synapses with the experimental data set. This analysis showed that compared with the randomly distributed synapses, the experimental data exhibited a larger fraction of branches at the extremes of the distribution, i.e., that have no TC synapses or a high TC synapse density (Figure 9D, Kolmogorov-Smirnov (KS) test, significant, p = 1.1 × 10−37). These findings therefore indicate that TC afferents do not have equal preference for all basal dendritic branches on L5 pyramidal neurons but, instead, are biased toward a subset of preferred dendritic branches.

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