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Cell-type specific innervation of cortical pyramidal cells at their apical dendrites

  1. Ali Karimi
  2. Jan Odenthal
  3. Florian Drawitsch
  4. Kevin M Boergens
  5. Moritz Helmstaedter  Is a corresponding author
  1. Department of Connectomics, Max Planck Institute for Brain Research, Germany
Research Article
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Cite this article as: eLife 2020;9:e46876 doi: 10.7554/eLife.46876

Abstract

We investigated the synaptic innervation of apical dendrites of cortical pyramidal cells in a region between layers (L) 1 and 2 using 3-D electron microscopy applied to four cortical regions in mouse. We found the relative inhibitory input at the apical dendrite’s main bifurcation to be more than 2-fold larger for L2 than L3 and L5 thick-tufted pyramidal cells. Towards the distal tuft dendrites in upper L1, the relative inhibitory input was at least about 2-fold larger for L5 pyramidal cells than for all others. Only L3 pyramidal cells showed homogeneous inhibitory input fraction. The inhibitory-to-excitatory synaptic ratio is thus specific for the types of pyramidal cells. Inhibitory axons preferentially innervated either L2 or L3/5 apical dendrites, but not both. These findings describe connectomic principles for the control of pyramidal cells at their apical dendrites and support differential computational properties of L2, L3 and subtypes of L5 pyramidal cells in cortex.

Introduction

The apical dendrites of pyramidal neurons, the most abundant cell type in the mammalian cerebral cortex (Cajal, 1899; DeFelipe and Fariñas, 1992), have been a key focus of anatomical (Cajal, 1899), electrophysiological (Stuart and Sakmann, 1994; Larkum et al., 1999) and in-vivo functional studies (Svoboda et al., 1997; Letzkus et al., 2011; Takahashi et al., 2016). Of particular interest has been the coupling of long-range top-down inputs converging at the apical tufts in layer 1 with synaptic inputs on the basal dendrites in large layer 5 pyramidal cells (Larkum et al., 1999; Larkum, 2013). Since almost all pyramidal cells from cortical layers 2, 3 and 5 (and some from layer 4) form their apical dendrite’s main bifurcation in this region, the layer 1/2 border in the upper cortex allows for synaptic convergence onto 80–90% of all cortical pyramidal cells (Larkman and Mason, 1990; Ito et al., 1998). Furthermore, the apical dendrites’ main bifurcation has been implied as a site of origin and control of regenerative electrical events in the distal dendrites (Helmchen et al., 1999; Larkum and Zhu, 2002; Larkum et al., 2004). Understanding the innervation profile of synaptic input onto apical dendrites in this peculiar part of the mammalian cerebral cortex was the goal of this study.

Results

We acquired four 3D-EM datasets from mouse somatosensory (S1), secondary visual (V2), posterior parietal (PPC) and anterior cingulate cortex (ACC) sized between 72 × 93 × 141 µm3 and 66 × 89 × 202 µm3 (Figure 1a, Figure 1—figure supplement 1, Table 1 in Supplementary file 1) at a voxel size of 11.24–12 × 11.24–12 x 28–30 nm3 using serial block-face electron microscopy (SBEM [Denk and Horstmann, 2004]). The image volumes were located at the border of layers 1 and 2, the site of the main bifurcation of apical dendrites from almost all pyramidal neurons of layers 2 to 5 (Figure 1a,b). To determine the layer origin of each apical dendrite (AD), we first identified those ADs with a soma in the image volume as L2 ADs, and the remaining ADs as ‘deeper layer’ (DL ADs), that is L3 and L5 ADs (Figure 1a). Synapses onto ADs were identified as targeting the shafts vs. the spines of ADs (for an identification of shaft-preferring axons as putative inhibitory vs. spine-preferring axons as putative excitatory, see below) without consideration of the symmetry of pre- and postsynaptic densities (Gray, 1959; Colonnier, 1968).

Figure 1 with 1 supplement see all
Complete synaptic input mapping of pyramidal cell apical dendrites (ADs) around their main bifurcation.

(a) Overview EM images indicating the location of 3D EM datasets in primary somatosensory (S1), secondary visual (V2), posterior parietal (PPC) and anterior cingulate (ACC) cortices relative to pia surface (solid line) and layer 1 / 2 border (dashed line). Schematic location of layer 2 (gray) and deep layer (orange) pyramidal neurons. (b) Reconstruction of all ADs contained in the ACC dataset (blue-green, n = 61 layer 2 and n = 152 deep layer ADs, respectively) and a subset of axons innervating them (n = 62, yellow-orange). Note that 80–90% of all pyramidal cells in cortex extend their apical dendrites into the L1/2 border region, allowing for massive synaptic convergence (https://wklink.org/8300). (c) Complete synaptic input maps of apical dendrite main bifurcations for deep layer (orange, S1: https://wklink.org/5413, V2: https://wklink.org/6059, PPC: https://wklink.org/9261, ACC: https://wklink.org/8408) and layer 2 (gray, S1: https://wklink.org/4049, V2: https://wklink.org/8965, PPC: https://wklink.org/8307, ACC: https://wklink.org/8695) pyramidal cells (example excitatory (spine, red spheres, https://wklink.org/9524) and putative inhibitory (shaft, blue spheres, https://wklink.org/2603) synapses, inset). (d) Box plot of putative inhibitory (blue crosses) and excitatory (red crosses) synapse densities per µm of AD shaft path length for L2 (n = 41, left) and DL ADs (n = 41, right) in S1, V2, PPC and ACC datasets. Wilcoxon rank-sum test, p < 10−6, 10−7 for excitatory and putative inhibitory densities, respectively. (e) Same as (d) reported separately per cortical region (n = 20 for S1, V2 and PPC, n = 22 for ACC). (f) Box plot of fraction of putative inhibitory synapses at the main bifurcation of deep (orange) and layer 2 (gray) ADs; individual ADs shown (crosses). (g) As (f) reported separately per cortical region. Asterisks indicate significance level of the Wilcoxon rank-sum test (*p < 0.05, **p < 0.01, ***p < 10−3, ****p < 10−4). Scale bars: 0.5 µm (inset in c).

Figure 1—source data 1

The density of excitatory and putative inhibitory synapses (Figure 1d, e).

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Figure 1—source data 2

The fraction of excitatory and putative inhibitory synapses (Figure 1f, g).

https://cdn.elifesciences.org/articles/46876/elife-46876-fig1-data2-v2.xlsx

Putative inhibitory to excitatory synapse ratio

We then identified all the synapses onto 82 ADs within 10 µm around their main bifurcation (Figure 1c, n = 6240 synapses total, of these n = 1092 shaft synapses, total dendritic length analyzed: 3.29 mm). The density of shaft synapses on DL ADs was 0.22 ± 0.02 synapses per µm dendritic shaft path length (mean ± SEM, n = 41, Figure 1d,e). Together with the substantial synaptic input to the spines of these ADs (Figure 1d,e, 2.16 ± 0.16 synapses onto spines per µm dendritic shaft path length), this yielded a fraction of 9.9 ± 5.1% (Figure 1f,g, mean ± SD, n = 41) shaft synapses at the main bifurcations of DL ADs in S1, V2, PPC and ACC cortex.

Next, we analyzed the synaptic input to L2 pyramidal cells’ ADs (Figure 1c–g). Here, unlike for DL ADs, we found substantial synaptic input to the shafts of the AD at the main bifurcation (Figure 1d,e, 0.42 ± 0.02 synapses per µm dendritic shaft path length, mean ± SEM, n = 41), with a fraction of 33.6 ± 17.9% (Figure 1f,g, mean ± SD, n = 41) of shaft synapses for S1, V2, PPC and ACC. Thus, L2 ADs receive about 3-fold more relative synaptic input at the shafts of their main bifurcation compared to L3 and L5 pyramidal cells (9.9% vs 33.6%, Wilcoxon rank-sum test, p < 10−11).

Shaft vs. spine synapses

For this analysis, we identified putative inhibitory versus excitatory synapses by their location on dendritic shafts versus spines (See Materials and methods, [Braitenberg and Schüz, 1998; Kubota et al., 2016]). To estimate the resulting rate of misidentification (i.e. of synapses onto shafts that were made by excitatory axons and synapses onto spines that were made by inhibitory axons, Figure 2a–c), we selected a subset of synapses onto shafts and spines of ADs and reconstructed the presynaptic axons. Then, we identified all other output synapses of these axons and determined the fraction of axonal synapses made onto spines (Figure 2a,b, Table 4 in Supplementary file 1; only single spine innervations were considered; analysis separately for synapses made within L1 and L2, Figure 2b). We found that, in fact, the preference of axons to either innervate shafts or spines of ADs was almost binary (Figure 2b): 91 of 92 axons seeded from the shafts of L2 ADs made at least 80% of their other output synapses again onto shafts (and 89 of 92 axons made at least 90% of their other synapses again onto shafts). Similarly, 34 of 35 axons seeded from spines at L2 ADs made at least 80% of their other synapses as primary spine innervations. Numbers for synapses seeded at DL ADs were comparable. This clear dichotomy supported the identification of spine-preferring axons as putative excitatory and shaft-preferring as putative inhibitory (Figure 2b, see also Motta et al., 2019). With the exception of axons innervating L5 slender tufted pyramidal cells, the resulting prediction accuracy of single synapses to predict the shaft or spine preference of the presynaptic axon was above 75%, with most innervations above 90% (Figure 2c). We used the ensuing correction factor for L5st neurons (Figure 2c) in the following.

Figure 2 with 1 supplement see all
Effect of synapse identity, size, dendrite diameter and double-innervated spines on synaptic composition of apical dendrites.

(a) Reconstruction of two axons seeded from synapses onto ADs (asterisks) and all the output synapses within the volume (red: spine, blue: shaft/non-spine). Note the stark difference in their innervation preference based on their seed location. Example spine (red arrows, 3: https://wklink.org/5224, 4: https://wklink.org/9411) and shaft (blue arrows, 1: https://wklink.org/8807, 2: https://wklink.org/3270) synapse EM micrographs (insets). (b) Histogram for the fraction of synapses made onto single-innervated spines. Axons seeded from synapses on deep (orange), L2 (gray), L3 (green), L5tt (magenta) and L5st (blue) ADs. Annotations are from layer 1 (n = 142, LPtA dataset) and layer 2 (n = 288, S1, V2, PPC and ACC datasets); probability density estimations and synapse identity threshold as solid and dashed lines, respectively. Note highly bimodal distribution allowing the clear distinction of axons into spine-preferring (excitatory) and shaft-preferring (putative inhibitory), with exception of axons innervating shafts of L5st ADs (these were corrected in the following, see c). (c) Scatter plot for accuracy in predicting presynaptic axon type (shaft- vs. spine-prefering) based on the location of synapse on AD (shaft vs. spine). (d) Size of synapses onto deep (n = 41, orange crosses) and layer 2 (n = 41, gray crosses) ADs. (e) Fraction of double-innervated spines in the main bifurcation annotations of deep (orange, n = 41) and layer 2 (gray, n = 41) neurons. Spine with putative inhibitory (blue arrow) and excitatory (red arrow) inputs (inset). (f) Sketches illustrating the synapse density normalized to surface area and length of AD and their measurement (upper panel). Box plot of the average diameter of layer 2 (gray, n = 41) and deep (orange, n = 41) AD around the main bifurcation in S1, V2, PPC and ACC datasets (lower panel, p < 10−4, Wilcoxon rank-sum test). (g) Box plot of putative inhibitory (blue crosses) and excitatory (red crosses) synapse densities per µm2 of AD surface area for L2 (n = 41, left) and DL ADs (n = 41, right). Wilcoxon rank-sum test, p < 10−6 for both densities. Asterisks indicate significance level of the Wilcoxon rank-sum test (not significant (n.s.): p > 0.05,*p < 0.05). Scale bars: 1 µm (inset in a,f).

Figure 2—source data 1

Dendritic spine vs. shaft innervation preference for AD-targeting axons (Figure 2b).

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Figure 2—source data 2

Prediction accuracy for dendritic shaft vs. spine innervation preference (Figure 2c).

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Figure 2—source data 3

Size of synapses onto ADs (Figure 2d).

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Figure 2—source data 4

Fraction of dendritic spines with double-innervation (Figure 2e).

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Figure 2—source data 5

Apical dendrite diameter and the synapse density normalized to surface area of ADs (Figure 2f, g).

https://cdn.elifesciences.org/articles/46876/elife-46876-fig2-data5-v2.xlsx

Synapse size, multiple spine innervations

We next investigated whether the observed difference in putative inhibitory synapse density for L2 versus DL ADs could be counteracted by differences in synaptic size (shown to be correlated to synaptic strength for excitatory synapses, [Cheetham et al., 2014; de Vivo et al., 2017; Holler-Rickauer et al., 2019, Figure 2d]). Synapse size onto spines was indistinguishable between L2 and DL ADs (Figure 2d), but synapses onto shafts of L2 ADs were slightly larger than those onto shafts of DL ADs (Figure 2d). Thus, if synapse size of shaft synapses was an indicator of synaptic strength, this would further enhance, not compensate the observed difference in putative inhibitory synapse density for L2 vs DL ADs.

A fraction of dendritic spines in cortex have been reported to be innervated by two or more synapses, with the secondary innervation often inhibitory (Kubota et al., 2007). A high rate of such multiple spine innervations could thus confound our results (Figure 1d–g). We measured the rate of multiple synapses onto spines (Figure 2e) and found it to be low (6.4 ± 0.9% vs 3.5 ± 0.4% for L2 vs DL ADs, mean ± SEM, p = 0.014, Wilcoxon rank-sum test). The difference in multiple spine innervations for L2 vs DL ADs would again further enhance, not reduce, the observed difference in putative inhibitory synapse density for L2 vs DL ADs.

Synaptic density per dendritic path length vs. surface area

We quantified synapse density per path length of the innervated dendritic shaft of the ADs (Figure 1). Differences in dendritic diameter between L2 and DL ADs could yield a differential effect on synaptic density per dendritic surface area for these innervations. It has been argued that areal synapse density is biophysically more relevant (since the input impedance of the dendrite is inversely correlated with its diameter [Rall and Rinzel, 1973; Rinzel and Rall, 1974; Bloss et al., 2016]). We therefore measured the diameter of ADs at their main bifurcation and found L2 ADs to be 21% wider than DL ADs (Figure 2f, Figure 2—figure supplement 1, diameter of 2.16 ± 0.06 µm vs 1.77 ± 0.04 µm for L2 vs DL ADs, respectively, mean ± SEM, n = 82, Wilcoxon rank-sum test, p < 10−4). L2 neurons, even though they had larger diameters, still received 59% higher absolute putative inhibitory synapse density per unit surface area compared to their DL counterparts (Figures 1d and 2g, compare to 89% density difference when normalized to shaft path length). The ratio of putative inhibitory to excitatory synaptic inputs (Figure 1) is not affected by these considerations.

Preference of presynaptic axons for types of apical dendrites

We next wanted to understand whether the presynaptic inhibitory axons that innervated ADs showed any innervation selectivity regarding the layer origin of the targeted pyramidal cells’ AD (Figure 3a–b). This was of special interest since the main bifurcations of all pyramidal cells reside in the same spatial region at the L1/2 border in cortex (Figure 3c, see also Figure 1b). We therefore reconstructed presynaptic inhibitory axons that had made at least one synapse onto a pyramidal apical dendrite shaft (Figure 3a), identified all other output synapses of these axons, and determined whether these were made again onto the same or different types of pyramidal cell ADs.

Preference of inhibitory axons for the type of postsynaptic pyramidal cell.

(a) Sketch illustrating two extreme innervation models for AD-targeting inhibitory axons: innervation could be selective for the type of AD (L2 vs L3/5 pyramidal cells, green), or indiscriminate for the type of AD (black). In the latter case, conditional dependence of targeting p(B|A) would be expected to be indistinguishable from overall probability of innervation p(B). (b) Conditional dependence of synaptic innervation p(B|A) shown as the mean probability of deep and layer 2 AD targeting (target ‘B’) given the target of the first synapse of an axon (target ‘A’, left panel). Probabilities reported as the average fraction of AD synapses onto each AD type. Box plot of AD fractional innervation for axons seeded from layer 2 (n = 92, gray crosses) and deep (n = 91, orange crosses) ADs (right panel), corresponding to the entries in the innervation matrix (left panel). P-values: Wilcoxon rank-sum test. Note that indiscriminate innervation can be revoked. (c) Distribution of main bifurcations along cortical depth (n = 41 per AD type; probability density estimates, lines), and distribution of axonal path length along cortical depth (n = 183 axons, of these n = 92 seeded at L2 (gray) and n = 91 seeded at DL ADs (orange)). Note that neither the pre- nor the postsynaptic targets are sorted along the cortical axis, excluding simple layering effects for the conditional innervation (b,c). Error bars indicate mean ± SEM over cortical region (S1, V2, PPC and ACC). (d) Mapping of axonal output onto subcellular targets. Error bars indicate mean ± SEM; asterisks: significance of bootstrapping test at p = 0.05 with Bonferroni correction. (e) Sketch illustrating innervation of an AD with multiple synapses (box). (f) Box plot for comparison of the average number of synapses per individual AD target for axons seeded from L2 (gray crosses) and deep (orange crosses) ADs. Wilcoxon rank-sum test, p = 0.49, 0.23 for L2 and deep ADs, respectively. (g) Conditional dependence of AD innervation p(B|A) where multiple innervation of same AD target were ignored (Compare to b). Probabilities indicate fraction of individual ADs targeted by axons seeded from L2 (first row) and deep (second row) ADs.

Figure 3—source data 1

Conditional innervation probability of sub-cellular structures by AD-targeting putative inhibitory axons (Figure 3b, d and Figure 4b, c).

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Figure 3—source data 2

The coordinate of analyzed AD main bifurcations (y-axis corresponds to the depth relative to pia, Figure 3c).

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Figure 3—source data 3

Distribution of AD-targeting axonal path length along the cortical depth in S1, V2, PPC and ACC datasets (Figure 3c).

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Figure 3—source data 4

Average number of synapses on individual AD targets by putative inhibitory axons (Figure 3f).

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Figure 3—source data 5

The distribution for the number of synapses on individual AD targets by putative inhibitory axons (Figure 3f, g).

https://cdn.elifesciences.org/articles/46876/elife-46876-fig3-data5-v2.xlsx

About 20% of synapses formed by each axon were again established onto apical dendrites (Figure 3b,d, n = 183, 20.3 ± 1.1%, mean ± SEM). AD innervation showed substantial conditional dependence on the type of AD the axon had been seeded from (Figure 3b), rejecting a model of indiscriminate inhibitory AD innervation: Axons seeded at DL ADs made 15.4 ± 1.6% of the remaining output synapses onto DL ADs (i.e. 71.2 ± 3% of all synapses onto ADs, mean ± SEM), but only 5.2 ± 0.8% onto L2 ADs (i.e. 28.8% of AD synapses, Figure 3b, n = 91, p < 10−8, Wilcoxon rank-sum test). Conversely, axons seeded at L2 ADs did not innervate their DL counterparts to a substantial fraction (Figure 3b, 4.1 ± 0.6%, n = 92); Rather, they innervated L2 ADs (15.9 ± 1.4%, i.e. 77.4% of their AD synapses). Since the paths of these inhibitory axons along the depth of the cortex were indistinguishable for those axons targeting deep and layer 2 ADs (Figure 3c, 18.1 vs. 16.7 mm, n = 183), this connectomic specificity far exceeded average random innervation determined by the trajectory of the presynaptic axons.

Contribution of multiple innervations to axonal target preference

Next, we investigated to what degree the innervation of individual apical dendrites by multiple synapses from the same inhibitory axon contributed to the establishment of axonal target preference for types of ADs (Figure 3e). Axons targeted ADs mostly through monosynaptic innervation regardless of the AD type (Figure 3f, 77% of connections made by one synapse; average of 1.32 ± 0.05 vs. 1.29 ± 0.04 synapses per AD target for axons seeded from L2 and DL ADs, respectively, mean ± SEM, Wilcoxon rank-sum test, p = 0.83). Thus, when evaluating the conditional innervation preference of L2 and DL ADs for presynaptic axons while disregarding multiple innervations of the same individual AD by the same presynaptic axon (using a binarized innervation, Figure 3g), the conditional axonal target preference was still substantial (Figure 3g, compare to Figure 3b, 70.8 ± 3.6% vs. 35.3 ± 4.2% individual L2 target fraction for axons seeded from L2 and DL ADs, respectively, mean ± SEM, n = 159, Wilcoxon rank-sum test, p < 10−7).

Comparative analysis of innervation preference between cortex types

Since we had acquired four 3D EM datasets from different parts of the cerebral cortex including primary sensory (S1), secondary sensory (V2), and higher-order (ACC, PPC) areas (Figure 4a), we then wanted to determine the variability or consistency of synaptic target preference across these cortical regions. We found remarkable quantitative consistency in innervation preference for axons seeded from L2 ADs (Figure 4b). The coefficient of variation (CV) of fractional innervation preference across cortical regions was 19.9%, 39.6%, 19.2% for reinnervation of L2 ADs, DL ADs and somata by L2 AD-seeded axons, respectively. For axons seeded at DL ADs, the CV of fractional innervation preference across cortical regions was 52.3%, 36.6%, 69.1% for reinnervation of L2 ADs, DL ADs and somata, respectively. Only reinnervation of L2 soma by deep-seeded axons showed significant variability across cortical regions (Figure 4c, MANOVA test followed by multiple one-way ANOVAs, p = 0.005). Note that all these axons had at least one synapse onto a shaft or spine of ADs, such that axons that innervated no shaft or spine of ADs were not included in this comparison.

Quantitative consistency in the innervation profile of inhibitory axons across four cortical regions.

(a) Sketch of the horizontal view of the mouse brain with location of cortical datasets used for comparison. See also Figure 1a. (b,c) Comparative analysis across cortical regions. Postsynaptic target specificity for axons seeded from (b) layer 2 ADs (n = 21, 20, 21, 30 for S1, V2, PPC and ACC, respectively) and (c) for axons seeded from deep layer ADs (n = 19, 20, 20, 32 for S1, V2, PPC and ACC, respectively). Note the high level of quantitative consistency of synaptic target fractions across cortices with the exception of somatic innervation in axons seeded from DL ADs. Error bars indicate mean ± SEM. *p < 0.05 MANOVA test followed by multiple One-way ANOVA with Bonferroni correction.

More detailed pyramidal cell type classification

We next wanted to determine whether the observed innervation differences were common to supragranular (L2/3) vs infragranular (L5) pyramidal cells (thus our data was possibly diluted by L3 pyramidal cells classified as ‘deep layer’), or whether the putative inhibitory innervation was in fact distinct between L2 and the other pyramidal cells (Figure 5). We used two datasets in which directly adjacent to the high-resolution EM data volume a low-resolution EM dataset had been acquired that extended to infragranular layers and allowed the following of ADs to their soma of origin (Figure 5a, datasets from PPC and LPtA cortex, Figure 5—figure supplement 1 for gallery of EM-based pyramidal cell reconstructions). In these datasets we could furthermore distinguish L2 pyramidal cells located directly at the L1-L2 border with a less prominent apical dendrite (L2 marginal neurons, L2-MNs, Figure 5a).

Figure 5 with 1 supplement see all
Synaptic input composition for L2, L3, and subtypes of L5 pyramidal cells at their main bifurcations.

(a) Complete skeleton reconstruction of apical dendrites contained within the high- and low-resolution volume of the PPC cortex dataset (PPC-2). Main bifurcation (black/red rectangles) and soma of origin (circles) is marked for L2 (gray, https://wklink.org/7762), L2MN (black, https://wklink.org/5352), L3 (green, https://wklink.org/7652), L5tt (magenta, https://wklink.org/3415) and L5st (blue, https://wklink.org/1435) neurons. (b) Sketch illustrating four morphological features of L5 neurons used for subtype classification. Insets show histograms of each feature for the L5tt (magenta, n = 7) and L5st (blue, n = 11) neurons. Note the differences between the two cell types. (c) Agglomerative hierarchical linkage tree (upper panel) and scatter plot of the first two principal components (lower panel) for (b). (d) Box plot for comparison of spine density between L5tt (magenta, n = 16) and L5st (blue, n = 17) neurons at their main bifurcation and distal apical tuft. (e) Fraction of putative inhibitory input synapses around the main bifurcation of apical dendrites from layer 2 (gray, n = 10), 2MN (black, n = 2, box plot shared with L2 group), 3 (green, n = 10), 5tt (magenta, n = 7), 5st (blue, n = 11) pyramidal cells (box plots) and density of excitatory (red crosses) and putative inhibitory (blue crosses) synapses at the main bifurcation (inset). Note clear distinction of synaptic input composition for L2 vs L3 and L5tt pyramidal cells at their main bifurcation. Kruskal-Wallis test, p < 10−4. (f) Scatter plot of synapse densities (left panels) and putative inhibitory fraction (right panel) of L5 neurons in relation to their thick-tuftedness (PC 1). Skeleton reconstruction of neurons indicated by numbers in the left panel demonstrate the relationship of PC 1 to AD tuft size (https://wklink.org/1293). Excitatory synapse density and putative inhibitory fraction have linear (Edensity=0.44×PC1+1.4, R2 = 0.74, p < 10−5) and non-linear relationships (Ifraction=0.043×PC1+0.130.58×PC1+1.40, R2 = 0.70, p < 10−7) to thick-tuftedness, respectively. Dashed lines in right panel indicate models using only linear excitatory (red), linear putative inhibitory (blue) or combined excitatory and putative inhibitory linear relationships (black).

Figure 5—source data 1

Morphological properties of L5 pyramidal neurons (Figure 5b, c, f).

https://cdn.elifesciences.org/articles/46876/elife-46876-fig5-data1-v2.xlsx
Figure 5—source data 2

Dendritic spine density for subtypes of L5 pyramidal neurons (Figure 5d).

https://cdn.elifesciences.org/articles/46876/elife-46876-fig5-data2-v2.xlsx
Figure 5—source data 3

More detailed cell-type comparison of the synapse density and fraction at the main bifurcation of ADs (Figure 5e).

https://cdn.elifesciences.org/articles/46876/elife-46876-fig5-data3-v2.xlsx

In order to also differentiate between L5 pyramidal cells termed ‘thick tufted’ and ‘slender tufted’ (Hübener and Bolz, 1988; Hübener et al., 1990; Manns et al., 2004), we used the observation that pyramidal cells with a clear ‘slender’ morphology (small tuft, few oblique dendrites, [Larkman and Mason, 1990]) had smaller soma diameters than those clearly identifiable as thick tufted (Figure 5b). In addition, the cortical depth of the main bifurcation (Staiger et al., 2016), number of oblique dendrites and diameter of the apical dendrite Groh et al., 2010; Narayanan et al., 2015 have been reported to differ between L5tt and L5st pyramidal cells. We therefore used these four features to cluster L5 pyramidal cells into L5tt and L5st (Figure 5c). The resulting subtypes differed in spine density (Figure 5d, 1.4 ± 0.24 vs. 0.59 ± 0.13 spines per µm path length for L5tt and L5st, respectively, Wilcoxon rank-sum test, p = 0.004).

When we then analyzed the fractional putative inhibitory synapse densities at the main bifurcation for L2, L3, L5tt and L5st pyramidal cells (Figure 5e), we found that, in fact, L2 pyramidal cells were unique in their high putative inhibitory synapse fraction. L3 pyramidal cells, to the contrary, showed the lowest putative inhibitory synapse fraction, comparable to L5tt pyramidal cells (putative inhibitory synapse fractions of 38.0 ± 4.9% vs 7.8 ± 0.8% vs 8.7 ± 1.1% vs 15.3 ± 3.0% for L2, L3, L5tt and L5st pyramidal cells, respectively, mean ± SEM, n = 40, Kruskal-Wallis test, p < 10−4, Post-hoc Tukey’s range test, p = 2.7 × 10−5 and 0.002 for L2 vs. L3 and L5tt, respectively).

The putative inhibitory synapse fraction in L5 pyramidal cells was strongly anti-correlated to the ‘thick-tuftedness’ of the L5 pyramidal cell (quantified as the first principal component in the four-parameter space used for L5tt vs L5st distinction above, Figure 5b,c,f). Interestingly, this effect was present although we had found that the shaft of L5st apical dendrites had substantial input from spine-preferring axons (Figure 2b–c, Table 4 in Supplementary file 1, 44% and 60% of shaft synapses onto L5st ADs were likely excitatory in L1 and L2, respectively), and we had corrected the synapse densities for L5st neurons accordingly (Figure 5e). The positive correlation between excitatory synapse density and the thick-tuftedness of L5 neurons was the main driver for the reduction in putative inhibitory fraction in L5tt neurons (Figure 5f, left panel, Linear fit: R2 = 0.74, p < 10−5). Using only the linear fit to excitatory synapse density and the average putative inhibitory synapse density, 55% variability in the putative inhibitory fraction of L5 neurons could be explained (Figure 5f, right panel, red dashed line). For comparison, a linear fraction model fitted to the data explained an additional 15% of variance in the putative inhibitory fraction (Figure 5f, right panel, black solid line, R2 = 0.70, p < 10−7, Linear fraction model, n = 4 parameters).

Two identified L2 marginal neurons (L2MN, with an AD that runs oblique to the pial surface, Figure 5a, [Larkman and Mason, 1990; Luo et al., 2017]) showed putative inhibitory synapse fractions at their main bifurcation that were consistent with that of other L2 neurons (Figure 5e, 53.2% and 27.8% putative inhibitory fraction for L2MN).

Effect of pyramidal cell type vs. distance to soma

The observed pyramidal-cell-type specific putative inhibitory innervation was based on a cell type classification that relied largely on the cortical layer position of the pyramidal cell’s soma (Figure 5a). Since main bifurcations were located approximately at the same cortical depth around the L1-to-L2 border (Figure 5a), the cell types differed substantially in their dendritic distance between the main bifurcation and the cell body (i.e. the length of their apical dendrite trunks). We therefore analyzed the relation between the fraction of putative inhibitory synapses at the main bifurcation and the distance of the main bifurcation to the soma and found a very strong distance-dependence (Figure 6a,c, R2 = 0.73 for single exponential fit, n = 81). This dependence was even evident within the population of L2 pyramidal cells, consistent with a possible synaptic specificity mechanism that is dependent on the distance to the cell body. Interestingly, this dependence was weaker for the absolute density of excitatory or putative inhibitory synapses, compared to their ratios (Figure 6b, R2 = 0.01, 0.56, respectively). The relationship was consistent for all pyramidal cell types except L5st (Figure 6a). The distance-dependence of the putative inhibitory input fraction was also found for apical dendrite locations before and after the main bifurcation within the L2 pyramidal cell population (Figure 6c).

Dependence of inhibitory input fraction at the main bifurcation of apical dendrites to the distance to soma.

(a) Relationship between distance of main bifurcation to soma and putative inhibitory fraction at the main bifurcation for L2 (n = 51, gray), L2MN (n = 2, black), L3 (n = 10, green), L5tt (n = 7, magenta) and L5st (n = 11, blue) ADs. Black line indicates single exponential regression (Imb =1.57e0.047 dMB+0.1, R2 = 0.73, p < 10−22). (b) Same as in (a) for putative inhibitory and excitatory synapse densities, respectively. Note tight distance dependence for the putative inhibitory input fraction (a), but not the absolute synapse densities (b). (c) Relationship between path distance to soma on the apical dendrite and the fraction of putative inhibitory synapses on L2 apical dendrites (n = 66, S1, V2, PPC, ACC, PPC-2 and LPtA datasets, n = 12,532 synapses). Each cross represents the putative inhibitory fraction (i/(i+e)) for a single apical dendrite within a 10 µm path length range (dAD) to cell body of origin (inset).

Figure 6—source data 1

Path distance between soma and main bifurcation (MB) with the corresponding synapse densities and fractions at MB (Figure 6a, b).

https://cdn.elifesciences.org/articles/46876/elife-46876-fig6-data1-v2.xlsx
Figure 6—source data 2

The fraction of putative inhibitory synapses along the apical dendrite of L2 neurons (Figure 6c).

https://cdn.elifesciences.org/articles/46876/elife-46876-fig6-data2-v2.xlsx

Putative inhibitory synapse fractions at the distal apical tuft

Finally, we analyzed the putative inhibitory and excitatory innervation of ADs towards their upper ends in L1 (Figure 7a,b, LPtA dataset). This was of special interest since the density of dendritic spines in hippocampal pyramidal cells had been previously found to decrease by about 5-fold towards the AD’s tip (Bannister and Larkman, 1995; Megías et al., 2001). We wanted to understand whether this was similar in cortical pyramidal cells, and whether this was accompanied by a drop (or increase) in putative inhibitory synapse density.

Figure 7 with 1 supplement see all
Inhibitory input fraction at distal apical tuft dendrites.

(a) Skeleton reconstruction of 14 L2, L3, L5tt and L5st ADs with all synapses mapped within the high-resolution EM image volume in layer 1 of LPtA (L2, L3, L5tt, https://wklink.org/9047) and PPC (L5st, https://wklink.org/8828) cortex (red: excitatory synapses, n = 3,812, cyan: putative inhibitory synapses n = 694). Note the difference in synapse densities (red/cyan sphere density) for layer 2, 3, 5tt, 5st ADs. (b) Box plot for fraction of putative inhibitory synapses at the distal apical dendritic site from L2 (n = 9 individual branches, gray crosses), L3 (n = 7, green crosses), L5tt (n = 9, magenta crosses) and L5st (n = 6, blue crosses) pyramidal cells and box plot for density of excitatory (red crosses) and putative inhibitory (blue crosses) synapses at the distal tuft (right panel). Note the clear distinction of synaptic input composition in the distal tuft for L2 vs. L3 vs. L5tt vs. L5st pyramidal cells (Kruskal-Wallis test, p < 10−4) mainly due to differences in excitatory synapse density. (c) Summary of distinct putative inhibitory input fraction at the main bifurcation and distal tufts of apical dendrites for four main classes of pyramidal cells in the cerebral cortex. Note that only L3 pyramidal cells show approximately homogeneous ratio of putative inhibitory and excitatory synaptic inputs along their apical dendrites. Error bars indicate mean ± SEM.

Figure 7—source data 1

The synapse density and fraction in the distal part of apical dendrites (Figure 7b).

https://cdn.elifesciences.org/articles/46876/elife-46876-fig7-data1-v2.xlsx

We mapped all input synapses onto 14 additional apical tuft dendrites (Figure 7a, n = 4506 synapses) and found that, first, the excitatory synapse density of L5tt pyramidal cells decreased 3-fold towards their distal tufts. This was not accompanied by a drop in putative inhibitory inputs, which remained almost constant in density (Figures 5e and 7b, Figure 7—figure supplement 1a–b). Thus, L5tt pyramidal cells received enhanced putative inhibitory input at their distal dendrites (Figure 7b, putative inhibitory input fraction 22.5 ± 1.7% at the distal tips, mean ± SEM, n = 16 dendritic segments, p < 10−3, Wilcoxon rank-sum test compared to MB). For L5st pyramidal cells, the putative inhibitory input fraction at their distal apical tufts was also higher than their main bifurcations, with about a third of their synapses putative inhibitory (Figure 7b–c, 15.3 ± 3% and 31.3 ± 1.8% putative inhibitory synapse fraction at main bifurcation and distal tips of L5st, respectively, mean ± SEM, n = 17, Wilcoxon rank-sum test, p = 0.005). Together, this demonstrated increased inhibition for all L5 pyramidal neurons at their distal AD.

For L2 pyramidal cells, to the contrary, putative inhibitory synapse density decreases ~2 fold towards the distal tuft (Figures 5e and 7b, 0.47 ± 0.02 vs 0.23 ± 0.03 putative inhibitory synapses per μm dendritic path, mean ± SEM, n = 62, Wilcoxon rank-sum test, p < 10−4) while the excitatory input increases by ~1.5 fold, yielding an about 3-fold reduced putative inhibitory input fraction towards their distal tufts (Figures 5e and 7b, 34.6 ± 2.5% putative inhibitory input fraction at main bifurcation vs 11.4 ± 1.4% at distal tufts, Wilcoxon rank-sum test, p < 10−4).

Finally, we found that L3 pyramidal cells show no substantial dependence of their synaptic input fractions on the position along the apical dendrite: they showed low putative inhibitory input fraction around their main bifurcation (Figure 7b–c, 7.8 ± 0.8%, mean ± SEM, n = 10, similar to the L5tt cells, Wilcoxon rank-sum test, p = 0.36), which only slightly increased towards their distal tufts (10.2 ± 0.9%, mean ± SEM, n = 7 tuft branches, similar to L2 cells, Wilcoxon rank-sum test, p = 0.84).

With this, we find that the pyramidal cells classified as L2, L3, L5tt and L5st show each distinct putative inhibitory innervation profiles along their apical dendrites, providing succinct possibilities for putative inhibitory control of excitatory inputs in the cortex (Figure 7c, Figure 7—figure supplement 1a–c) and emphasizing the need to consider L2 and L3 pyramidal cells (and subtypes of L5 pyramidal cells) as separate entities.

Discussion

We obtained a quantitative connectomic input map of pyramidal cell apical dendrites in several areas of the mouse cerebral cortex. We found distinct profiles of putative inhibitory to excitatory synaptic innervation ratio for all types of pyramidal cells (Figure 7): L2 pyramidal cells received substantial inhibition at their apical dendrites’ main bifurcation, which decreased about 3-fold towards the tips of the apical dendrites; in thick-tufted layer 5 pyramidal cells, this profile was inverted with a decreased fraction of putative inhibitory inputs at the main bifurcation. L5 slender-tufted cells followed the L5tt profile but received about 50% higher putative inhibitory input fraction overall. L3 pyramidal cells were the only cell type to show a homogeneous putative inhibitory input across upper cortex of about 10%. At the level of axons, putative inhibitory innervation exhibited connectomic specificity with axons preferring the innervation of either L2 or deeper-layer pyramidal cells (Figure 3). These data indicate a unique innervation profile for each pyramidal cell type, and point to a high variance of the cell-type specific ratio of inhibition and excitation in the cortex. Our findings were remarkably consistent for the investigated cortices (S1, V2, PPC and ACC, Figure 4).

Distinction of excitatory vs inhibitory synapses

For the distinction of excitatory vs. inhibitory synapses in single high-resolution EM images, the symmetry of pre- and postsynaptic densities and the properties of the synaptic cleft have been developed as criteria (Gray, 1959Colonnier, 1968). In large-scale 3D EM data, it is possible to image and analyze extended stretches of axons and assign their likely inhibitory vs. excitatory characteristic based on the target distribution of their synapses. This is especially fruitful when for the acquisition of large image volumes, compromises in in-plane image resolution have to be made as is the case for SBEM (insets in Figure 1c; see also [Staffler et al., 2017]). To calibrate the target bias of axons for innervating shafts vs. spines of dendrites, we mapped the preference of AD-targeting axons to innervate dendritic shaft and spines (Figure 2a–c), and found in all but one case clearly distinct axonal populations with almost exclusive targeting of shaft vs. spines. These axonal preferences appear even more distinct than the axonal preferences recently reported in L4 of mouse S1 cortex (Motta et al., 2019).

Synaptic vs. non-synaptic inhibition

In physiological experiments, evidence for a strong effect of GABA on regenerative activity in apical dendrites of thick-tufted L5 cells via non-synaptic GABA-B receptors had been reported (Pérez-Garci et al., 2006; Oláh et al., 2009; Pérez-Garci et al., 2013; Abs et al., 2018). Together with our evidence on low rates of synaptic putative inhibitory innervation at the main bifurcation of these pyramidal cells, this may indicate that L5tt pyramidal cells are controlled by overall and slow inhibition at their main bifurcations, but specific synaptic innervation at their distal dendrites, while L2 pyramidal cells receive specific synaptic inhibition at their main AD bifurcations. Whether L3 pyramidal cells share the electrical properties of L5 pyramidal cells is still controversial (de Kock and Sakmann, 2009; Barth and Poulet, 2012). Their synaptic input composition at the main bifurcation as shown here would indicate commonality; but this could be counteracted by different distribution of active transmembrane conductances (Waters et al., 2003; Ledergerber and Larkum, 2012). Their distinct putative inhibitory input profile towards the tips of the apical dendrites may suggest that they allow for specific computations not shared by L2 or L5 pyramidal cells.

Distal inhibitory domains

Spatial distribution of excitatory and inhibitory synapses on the dendritic surface has substantial effects for the integrative properties and output of neurons (Rall, 1959; Polsky et al., 2004; Katz et al., 2009). Previous studies in hippocampal pyramidal cells had reported a substantial increase in inhibitory synapse fraction towards the distal tuft dendrites which is driven mainly by a drop in excitatory synapse density (Bannister and Larkman, 1995; Megías et al., 2001; Bloss et al., 2016). We find that L5 pyramidal cells in cortex exhibit a similar distal putative inhibitory input domain. Theoretical work indicated that this is potentially a very powerful inhibitory effect (Gidon and Segev, 2012). This distal inhibitory domain, however, is absent in the distal tufts of L2 and L3 pyramidal neurons.

Distinction of L2 vs L3 pyramidal cells in cortex

Importantly, our data shows a striking specialization of L2 vs L3 pyramidal cells, which has so far evaded analysis (for example, [Iascone et al., 2018]). Only L3 (and to some degree L5st), but not L2 or L5tt pyramidal cells, keep a roughly even putative inhibitory to excitatory synaptic input ratio. Recent work using intracellular Gephyrin tagging for the detection of inhibitory synapses and counting of spines for excitatory synapses in LM (Iascone et al., 2018) underestimated the rate of spines by about ~20% compared to our complete 3D EM based sampling, resulting in an overestimation of the inhibitory-to-excitatory input fraction for L3 pyramidal cells (see also [Chen et al., 2012]). Our results emphasize the importance of separately analyzing L2 and L3 (and subtypes of L5) pyramidal cells in cortex (Figures 1, 5 and 7, see also [Meyer et al., 2011]).

Conclusion

In conclusion, our findings describe non-random connectivity at the apical tuft input domain of pyramidal neurons in cortex. They imply unique innervation patterns for the apical dendrites of L2 vs L3 vs L5 pyramidal cells yielding highly variable and cell-type specific putative inhibitory-to-excitatory synaptic input compositions, and suggest differential modes of inhibitory operation for the main classes of pyramidal cells in the cortex.

Materials and methods

Animal experiments

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All experimental procedures were performed according to the law of animal experimentation issued by the German Federal Government under the supervision of local ethics committees and according to the guidelines of the Max Planck Society. The experimental procedures were approved by Regierungspräsidium Darmstadt, under protocol ID V54 - 19c20/15 F126/1015 (LPtA, PPC-2) or V54 – 19 c 20/15 – F126/1002 (V2, PPC, ACC). The S1 sample was prepared following experimental procedures approved by Regierung von Oberbayern, 55.2-1-54-2532.3-103-12.

S1, LPtA sample preparation

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The cortical tissue processing, 3D electron microscopy and data alignment for S1 and LPtA samples were described previously in Berning et al. (2015) and Drawitsch et al. (2018), respectively. V2, PPC, ACC and PPC-2 samples were processed as follows.

Transcardial perfusion

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Adult (P56 – 57) wild type mice (C57BL/6J) were injected with general analgesia (0.1 mg/kg buprenorphine (Buprenovet, Recipharm, France) and 100 mg/kg Metamizol (Metamizol WDT, WDT, Germany)). Next, animals were anesthetized by inhalation of isoflurane (3–3.5% in carbogen) and were perfused transcardially using 15 ml of cacodylate buffer (150 mM, Serva, Heidelberg, Germany, pH = 7.4) followed by 30 ml of fixative solution at a flow rate of 10 ml/min. The fixative solution was 2.5% PFA (Sigma-Aldrich, Germany), 1.25% glutaraldehyde (Serva) and 0.5% CaCl2 (Sigma-Aldrich) in 80 mM cacodylate buffer (pH = 7.4). The animal was decapitated and the skull was removed to expose the brain. The head was next submerged in fixative solution overnight at 4°C.

Cortical region targeting and tissue extraction

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First, a 600 µm coronal slice containing the region of interest was acquired using a vibrating microtome (Microm HM 650V, Thermo Fisher Scientific, USA) guided by a reference atlas (Franklin and Paxinos, 2008). Next, we used a biopsy punch (1 mm in diameter, KAI medicals, USA) to extract a cylindrical piece of cortex containing layers 1–3 (Figure 1—figure supplement 1). This tissue was then incubated for 3–4 hr (V2, PPC and ACC) or overnight (PPC-2) in cacodylate buffer. The PPC-2 sample was also subjected to confocal laser scanning light microscopy as previously described (Drawitsch et al., 2018).

En-bloc sample preparation for 3D electron microscopy

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Samples were prepared for serial block-face electron microscopy following a slightly modified en-bloc staining method as described in Hua et al. (2015). In short, cortical tissue was rinsed in cacodylate buffer for 30 min before any staining material was applied. Next, it was transferred into 2% OsO4 (Serva, Germany) in cacodylate buffer for 90 min. The sample was then treated with 2.5% ferrocyanide (Potassium hexacyanoferrate trihydrate, Sigma-Aldrich, Germany) in cacodylate buffer for 90 min and 2% buffered OsO4 for 45 min. We then rinsed the tissue in cacodylate and ultrapure water (Biochrom, Germany) for 30 min each. Osmium content of the sample was amplified by treatment with saturated aqueous thiocarbohydrazide (TCH, Sigma-Aldrich, Germany) and 2% aqueous OsO4 for 60 and 90 min, respectively. The sample was moved to 2% uranyl acetate (Serva) solution for overnight incubation at 4°C. The following day, the tissue, still in uranyl acetate, was warmed to 50°C for 120 min in an oven (Memmert, Germany). This was followed by incubation in lead aspartate at 50°C for 120 min. The lead aspartate solution was prepared by dissolving 0.066 g lead nitrate (Sigma-Aldrich, Germany) in a 10 ml 0.03 M aspartic acid (Serva, Germany) solution and adjusting the pH to 5.0.

Next, the cortical tissue was dehydrated by incubation in 50–100% ethanol gradient (Serva, Germany). This was followed by at least three 20–45 min incubation steps in pure acetone (Serva, Germany). The sample was then transferred to a 1:1 mixture of acetone and Spurr’s resin (4.1 g ERL 4221, 0.95 g DER 736 and 5.9 g NSA, 113 µl DMAE, Sigma-Aldrich, Germany) for 3–4 hr with slow/no rotation. Tubes were opened to allow for acetone evaporation overnight (V2, PPC and ACC samples). At this stage, the PPC-2 sample was transferred to a 3:1 mixture of Spurr’s resin and acetone instead. The following day, the infiltration process continued for two 3 hr (PPC-2) or one 6 hr (V2, PPC, ACC) incubation steps in pure Spurr’s resin mixture. The tissue was then transferred to a flat-embedding mold and cured at 70°C for at least 48 hr. Specific time and temperature of dehydration and embedding steps are detailed in Table 2, Supplementary file 1.

Note that all procedures were performed at room temperature in 2 ml reaction tubes (Eppendorf, Germany) unless stated otherwise. In addition, the initial incubation steps (until dehydration) were performed with the aid of an automatic microwave tissue processor (Leica EM AMW, Leica, Germany) for V2, PPC, and ACC samples. Finally, treatment steps were interleaved with two 30 min washing steps in ultrapure water (from TCH step until dehydration initiation).

Serial block-face electron microscopy (SBEM)

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The samples were excised from the resin block and mounted on an aluminum pin using epoxy glue (Uhu plus schnellfest, Uhu, Germany). They were then trimmed to a block-face area of ~750 µm x 750 µm using a diamond head trimming machine (Leica EM TRIM2, Leica, Germany). In addition, tissue was sputter coated with 100–200 nm of gold (Leica ACE600 Sputter Coater, Leica, Germany) to increase conductivity and reduce charging artefacts.

The SBEM microtome (courtesy of W. Denk) was fit inside the door of a scanning electron microscope (Table 1 in Supplementary file 1, FEI, Thermo Fisher Scientific, USA). The microtome and microscope were controlled using custom written software during the volume imaging process; focus and stigmation were corrected manually (V2, PPC, ACC and LPtA) or using custom written auto-correction routines (S1, PPC-2). The region of interest was imaged using overlapping image tiles and cutting direction was along the tangential (S1, V2, PPC, ACC, PPC-2) or radial (LPtA) axes of cortex. The region of interest was targeted to an area close to layer 1/2 border (S1, V2, PPC and ACC, Figure 1a). The final imaged volume and the nominal voxel size is detailed in Table 1, Supplementary file 1. Note that the LPtA and PPC-2 datasets were adjacent to datasets extending to the middle of layer 5 imaged at lower resolution (22.48 × 22.48 × 30 and 44.96 × 44.96 × 120 nm3 for PPC-2 and LPtA, respectively).

Image alignment

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The alignment for PPC, V2 and ACC dataset was done using custom-written MATLAB (Mathworks, USA) routines. We used cross-correlation or speeded-up robust feature (SURF) detection in the overlap region to measure the relative shift between image patches. These patches were full image tiles (V2, ACC) or tile sub regions (PPC, 256 × 256 pixels). The position of each patch was then globally optimized using least-square regression. The image volume was then partitioned into 1024 × 1024 × 1024 voxel blocks and written into webKnossos (Boergens et al., 2017) three-dimensional format. The dataset was then transferred to the data store accessible by webKnossos for in-browser neurite skeleton reconstruction and synapse annotation.

The PPC-2 dataset was aligned using the affine alignment method from Scheffer et al. (2013). The routines were modified to give sub regions of image tiles unique affine transformations. In addition, methods to exclude featureless blood vessel and nuclei were improved.

Reconstruction and synapse annotation analysis

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The skeleton reconstruction and synapse annotations were saved as a NML (XML-based format) file. They were subsequently parsed into a MATLAB (release 2014b-2019a) class with node and edge list properties using custom-written C++ routines. Each node had accompanying attributes, such as, comment string and coordinate. These attributes were used to extract different features of the annotation as described in the following sections.

Apical dendrite (AD) definition and classification

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Apical dendrites (ADs) were identified based on their radial direction and diameter (~1–3 µm). The soma morphology and axon initial segment direction (towards white matter (WM)) of candidate pyramidal cells were examined where possible.

Within the S1, V2, PPC and ACC datasets, ADs were classified depending on the existence of soma in the image volume (Figure 1a). ADs with soma in the image volume were classified as layer 2 (L2) and other apical dendrite were classified as deep layer (DL or L3/5) ADs.

The depth of the apical dendrite’s source soma relative to pial surface was used to differentiate layer 2, 3 and 5 cells in the LPtA and PPC-2 datasets that were contained in layers 2–5 (Table 5 in Supplementary file 1). Note that, LPtA and PPC cortical regions do not possess a prominent layer 4 (Kolb and Walkey, 1987).

The difference between nominal and actual cutting thickness in the LPtA dataset was resulting in apparently ellipsoid somata (compressed along the cutting axis). To obtain an accurate estimate of soma depth relative to pial surface, the section thickness was corrected (by a factor of 1.49), assuming soma dimensions to be similar in-plane and along the cutting direction.

The main bifurcation of apical dendrites was defined as the branching point with two daughter branches of similar thickness and branching angle (resulting in a ‘Y’ shape, Figure 1c).

We used the ACC dataset to reconstruct all ADs (Figure 1b, blue-green). They were detected by examining the border of dataset facing WM for deeper layer ADs (n = 152) and by identifying all L2 pyramidal neurons contained for L2 ADs (n = 61). The AD locations were used as seed points for manual annotation ignoring spines.

Identification of layer 2 marginal neurons (L2MN) and slender-tufted L5 (L5st) ADs

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The AD morphology was reconstructed for all ADs investigated to identify the type of pyramidal neuron it belongs to (Figure 5a, Figure 5—figure supplement 1a). Next, we set out to find the less frequent subtypes for L2 and 5 neurons.

We used the orientation of the apical dendrite to find layer 2 marginal neurons that have an oblique AD which runs parallel to pial surface (Larkman and Mason, 1990; Luo et al., 2017; Figure 5a, Figure 5—figure supplement 1a, black reconstructions).

We also found apical dendrites of L5st neurons (Figure 5a, Figure 5—figure supplement 1a, blue reconstructions) based on the difference in their soma diameter (Larkman and Mason, 1990). We also measured their AD diameter, number of oblique dendrites and depth of main bifurcation to perform a hierarchical clustering. Using the correlation of the actual distance between clusters and the height of their link in the hierarchical tree, we found the average cosine distance to be the most consistent metric (one minus cosine of the angle between points in the four-dimensional morphological feature space). We found two clusters containing L5tt (n = 7) and L5st (n = 11) neurons (Figure 5b,c). Furthermore, we performed principal component analysis of the L5 morphological features. Since the first principle component (PC 1) correlated with the thick-tuftedness of L5 neurons, we investigated the linear relationship between PC 1 and synapse densities. Additionally, we used the fitted linear models to synapse density to predict the putative inhibitory fraction (Figure 5f, right panel, dashed lines). For comparison, we fitted a non-linear model (fraction of two linear expressions) to the putative inhibitory fraction (Figure 5f, right panel, solid line). We also compared the spine density between L5tt and L5st neurons at their main bifurcation and distal apical tuft (Figure 5d).

We approximated the soma volume using an ellipsoid by measuring the diameter along the three Cartesian dimensions of the dataset. We then calculated the diameter of the sphere that has an equivalent volume. To measure AD diameter, we determined the diameter of the sphere that has an equivalent area to the ellipse approximation of the AD trunk cross-section about 50 μm from the soma.

Complete synaptic input mapping of apical dendrites

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Apical dendrites and their associated spines were skeleton reconstructed and the synapses on their shaft and spines were annotated either within a bounding box of size 20 × 20 × 20 µm3 usually around the main bifurcation (Figures 1c–g, 5e, 6a and 7a–b (L5st group), n = 20 for S1, V2, PPC, n = 40 for PPC-2, n = 22 for ACC) or throughout the dataset (Figure 7a (except for the L5st group), Figure 7—figure supplement 1a, n = 11 for LPtA, n = 6 for ACC, n = 4 for V2, PPC and ACC) by a neuroscientist expert annotator (JO or AK).

Synapses were identified within the SBEM data based on the presence of vesicle cloud and postsynaptic density as described previously (Schmidt et al., 2017). We also annotated spine neck synapses and double innervations of spine heads by two axonal boutons (Figure 2e; Kubota et al., 2007). Shaft synapses, synapses on spine necks and secondary spine innervations were counted as putative inhibitory synapses and primary spine innervations were counted as excitatory synapses (Figures 1, 5 and 7) with the exception of L5st neurons (Figures 5e and 7b, uncorrected measurements in gray, Also see section: Identity estimation for shaft and spine synapses). All synapse annotations were validated by an additional expert annotator, and only synapses where annotators agreed were used for analysis.

The fraction of putative inhibitory synapses was defined as the number of putative inhibitory synapses divided by the total number of synapses (Figure 1f,g, Figures 5e and 7b). The putative inhibitory and excitatory synapse density was calculated by dividing the number of synapses by the path length of the apical dendrite shaft (Figure 1d,e, insets in Figures 5e and 7b). Shaft path length was measured by removing the spine necks from the skeleton reconstructions and summing the lengths of the remaining edges. Note that putative inhibitory input was measured separately for each distal tuft branch in the LPtA dataset resulting in 9, 7, 9 and 6 dendritic segments for L2, 3, 5tt and 5st cells, respectively (Figure 7a,b).

Apical dendrite diameter and synapse density per unit surface area

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Apical dendrite diameter was measured every ~2–3 µm along the path of each dendrite to get an average diameter (Figure 2f). The average diameter in combination with the path length of the dendrite was used to calculate the surface area of the dendrite (AD surface = π x path length x average diameter). We then calculated the synapse density per unit surface of the dendrite by dividing each synapse count by the surface area of the AD (Figure 2f–g).

Inhibitory input fraction mapping in upper cortex

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We first estimated the approximate location of each dataset relative to pia (Figure 1a, 125, 215, 170, 110, 10 and 20 µm for S1, V2, PPC, ACC, PPC-2 and LPtA, respectively) based on their position in coronal overview images and transformed all reconstructions into a common coordinate system (Figure 7—figure supplement 1a). Next, we partitioned these reconstructions (n = 141, total AD shaft path length = 14.1 mm) into virtual 100 µm thick cortical tangential sections. This process resulted in dendritic segments at each depth bin. We then combined synapse counts and path lengths from all segments within each virtual cortical section to calculate the total putative inhibitory fraction and synapse densities (lines in Figure 7—figure supplement 1b–c). We also used the 95% bootstrap confidence interval to estimate the dependence of the total average on the sample composition (shades in Figure 7—figure supplement 1b–c, n = 10,000 bootstrap samples).

Soma, main bifurcation distance effect on synapse composition at the main bifurcation

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We annotated the apical trunk connecting the soma to main bifurcation within the high- and low-res EM data volumes for the L2 (n = 51, S1, V2, PPC, ACC, PPC-2), L2MN (n = 2, PPC-2), L3 (n = 10, PPC-2), L5tt (n = 7, PPC-2), L5st (n = 11, PPC-2) pyramidal cells and considered the path length of these annotations as the soma to main bifurcation distance (Figure 6a–b).

Inhibitory fraction along the AD of L2 pyramidal neurons

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We binned the excitatory and putative inhibitory synapses based on their path distance to soma in the L2 pyramidal ADs (Figure 6c, n = 66 L2 dendritic segments, n = 12,532 synapses, bin size = 10 µm). This allowed us to measure the fraction of putative inhibitory synapses as a function of dendritic path distance to the soma (10–330 µm distance range). Soma distance bins with less than four synapses were merged to their immediate neighbor that contained at least four synapses. This was to avoid extreme values introduced by computing the putative inhibitory ratio in a bin with low synapse count.

Identity estimation for shaft and spine synapses

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A random subset of axons targeting shaft or spine of ADs were reconstructed (Figure 2a–c, n = 142 and 288 for datasets in layers 1 and 2, respectively). The postsynaptic targets were identified for at least six additional targets of each axon if possible. Next, we determined whether the postsynaptic target was single-innervated spine or shaft/non-spine (this included shaft, spine neck, double-innervated spine and soma). We then plotted the histogram (bin size = 0.1) and the probability density estimate (bandwidth = 0.0573) of single-innervated spine innervation fraction for axons seeded from shaft or spine of ADs within layers 1 and 2 (Figure 2b).

We then classified axons into excitatory and putative inhibitory by using a 50% threshold on the single-innervated spine innervation fraction (Figure 2b, dashed lines). Axons with majority of their synapses onto single-innervated spines were counted as excitatory and the putative inhibitory axons had less than half of their synapses on single-innervated spines. Next, we calculated the fraction of excitatory input on each structure (Table 4 in Supplementary file 1). Subsequently, we corrected synapse densities and fraction of putative inhibitory synapses based on the excitatory synapse fraction on each structure. We then compared the synaptic measurements before and after synapse identity correction (Figure 2c). Note that double-innervated spines were always assumed to have one excitatory and one inhibitory component and were not included in the correction procedure. Finally, we used the corrected synapse density/fractions for the L5st group due to large spine-preferring input fraction onto their dendrite shafts (Figures 5e and 7b, gray crosses are measurements before correction).

Synapse size estimation

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A random subset of shaft and spine (n = 41 per AD type) synapses were used to determine the synaptic interface area (Figure 2d, S1, PPC, V2 and ACC). For this, an expert annotator placed two edges along the longest dimension of the synapse and its approximate orthogonal direction. These two edges were used as minor and major axes of an ellipse to estimate the contact area (area=π*semi-major axis *semi-minor axis).

Distribution of main bifurcations and axonal paths along upper cortex

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We obtained the main bifurcation depth relative to pia by transforming the datasets into a common coordinate system as described above (n = 82, Figure 1c, main bifurcation annotations). Next, we used this to create a histogram (bin size of 20 µm) and probability density estimate (bandwidth: 25 µm) along the cortical depth (Figure 3c, left panel) for main bifurcation densities. Note that this is only a subset of main bifurcations within each image volume (See Figure 1b).

We used these datasets to also obtain a density estimate for inhibitory axonal paths. For this, we removed the nodes used for marking synapse locations in the inhibitory axonal reconstructions and computed the average fraction (across S1, V2, ACC and PPC datasets) of the remaining axonal path within 20 µm tangential cortical slices (Figure 3c, right panel).

Conditional innervation probability of inhibitory axons

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We selected a random subset of putative inhibitory synapses from layer 2 (n = 21, 20, 21, 30 for S1, V2, PPC and ACC) and deep layer (n = 19, 20, 20, 32 for S1, V2, PPC and ACC) ADs. An expert annotator (JO or AK) then reconstructed the axons throughout the dataset. We also annotated all the other synapses and postsynaptic partners for each axon. In addition, reconstructions were checked by another expert annotator (JO or AK) for morphological irregularities such as sharp branching angles and untraced endings.

The postsynaptic targets were categorized into one of the following: layer 2 or deep layer apical dendrite (shaft and spine of trunk and primary branches), shaft of other dendrites, single- or double-innervated (including neck targeting) spine, layer 2 cell body, axon initial segment (AIS) or glia. The innervation fraction of an axon was computed by dividing the number of synapses for each specific target by the total number of synapses of that axon (the seed synapse was excluded). The innervation fractions were averaged for each AD seed type (Figure 3b,d). Each dataset average was also computed separately (Figure 4b–c).

Multiple innervation of an AD by inhibitory axons

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To measure the frequency of multiple innervation of an AD by inhibitory axons used in our investigation of axonal specificity, we annotated all the cases were such multi-innervation happened (Figure 3e). We then measured the average number of synapses on each distinct AD target (Figure 3f). Axons with no synapses on a L2 (n = 18, 46 for L2 and DL seeded axons, respectively) or DL AD (n = 48, 24 for L2 and DL seeded axons, respectively) were excluded since averages are not defined on empty sets. To understand the effect of multiple innervation on AD type preference, we calculated the average fraction of AD targeting disregarding multiple innervation of the same target for axons seeded from either AD type (Figure 3g).

Visualization of neurites and their synapses

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To visualize the surface of the main bifurcation of PPC and S1 apical dendrites and axons, we segmented the image volume using SegEM (Berning et al., 2015) and collected all the segments comprising the dendritic shafts. The volume data was then imported to MATLAB, binarized and smoothed using a (9 × 9 × 9) voxel Gaussian convolution kernel with standard deviation of 8 voxels. The isosurface was then constructed at a threshold of 0.2 (Figure 1c).

Volume of the AD shaft at main bifurcation in V2 and ACC datasets were generated by tracing the outline of the dendrites using the volume tracing mode in webKnossos and 3D data was processed in a similar fashion (Figure 1c).

Skeleton reconstructions of axonal (Figure 2a) and dendritic paths (Figures 1b,c, 5a and 7a, Figure 5—figure supplement 1) were represented by tubes. In addition, spheres were added to represent input (Figures 1c and 7a, Figure 7—figure supplement 1a) and output (Figure 2a) synapses.

The EM ultra-structure of an AD, synapses and cell bodies was demonstrated by electron microscopy images (PPC and ACC datasets) of their cross-sections (Figures 1c and 2a,e,f).

The visualizations were generated using MATLAB (mathworks) and Amira (Thermo Fisher Scientific, USA).

Statistics

Significance of difference between L2 and DL AD excitatory and putative inhibitory synapse densities (Figures 1d,e and 2g), putative inhibitory fractions (Figure 1f,g), synapse size (Figure 2d), double innervation of spines (Figure 2e), AD diameter (Figure 2f), AD innervation fraction (Figure 3b) and L5 subtype comparisons (Figure 5d) were tested using the non-parametric Wilcoxon rank-sum test. In addition, the rank-sum test was used to test for the difference of putative inhibitory fraction and excitatory/putative inhibitory synapse densities between the main bifurcation and the distal tuft area for each pyramidal cell type (Figure 7c). Finally, the difference in the putative inhibitory fraction between L2 (with L2MN), 3, 5tt and 5st apical dendrites (n = 4 independent samples) at the main bifurcation and on the distal tufts were tested using the non-parametric Kruskal-Wallis test to account for the small sample sizes (n = 12, 10, 7, 11 main bifurcations and n = 9, 7, 9, 6 distal tuft branches for L2, 3, 5tt, 5st, respectively. Figures 5e and 7b) followed by Tukey’s honestly significant difference post-hoc test.

To identify significant differences in the axonal innervation fractions, a bootstrapping test was used. The specificities of the two axonal groups were concatenated and 10,000 bootstrap resamples were drawn to match the number of axons seeded from L2 and DL ADs. The mean difference in each bootstrap resample between L2 and DL groups was compared to the sample mean difference. The p-value was defined as the fraction of bootstrap resamples which had a value more extreme compared to the sample mean. The significance threshold was set to 0.05 with Bonferroni correction for eight comparisons (Figure 3d).

We used a multivariate analysis of variance (MANOVA) test to measure the effect of cortical region on inhibitory axonal innervation probability for six postsynaptic targets (AD, soma, spine and shaft of other dendrites). Axon initial segment (AIS) and glia innervation fractions were excluded since they contained few synapses and resulted in singular matrices when MANOVA test was applied (0.8%, 0.04% of total synapses, respectively). The null hypothesis states that the differences between the mean fractional innervations of putative inhibitory axons in S1, V2, PPC and ACC is statistically insignificant. We followed the MANOVA test when the null hypothesis was rejected with multiple comparisons using Bonferroni corrected one-way ANOVA tests (Figure 4b,c). We then determined the target with differential fractional innervation between different cortical regions.

To investigate the relationship between synaptic composition at the main bifurcation and its distance to soma, single-term exponential models (with offset) of the form y=c+a*ebx were fit to the data from layers 2–5. The coefficient of determination (R2) was computed as a goodness of fit measure. We also used R2 to compare models explaining the relationship between thick-tuftedness (PC 1 of the four dimensional L5 neuron morphological feature space) and the putative inhibitory fraction at the main bifurcation. To understand the relationship between morphology and synapse density of L5 neurons, we fitted a linear model to individual synapse densities and a linear fraction model (form: ax+bcx+d) to their putative inhibitory fraction.

Data availability

All 6 datasets are available for browsing at webknossos.org using the following links. S1: https://wklink.org/8732 V2: https://wklink.org/9812 PPC: https://wklink.org/1262 ACC: https://wklink.org/6712 LPtA: https://wklink.org/8912 PPC-2: https://wklink.org/6347 All software used for analysis is available at https://gitlab.mpcdf.mpg.de/connectomics/apicaltuftpaper (copy archived at https://github.com/elifesciences-publications/apicalTuftPaper) under the MIT license.

The following data sets were generated
The following previously published data sets were used

References

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    Textura Del Sistema Nervioso Del Hombre Y De Los Vertebrados: Estudios Sobre El Plan Estructural Y Composición Histológica De Los Centros Nerviosos Adicionados De Consideraciones Fisiológicas Fundadas en Los Nuevos Descubrimentos
    Moya N.
  2. Book
    1. Franklin K
    2. Paxinos G
    (2008)
    The Mouse Brain in Stereotaxic Coordinates
    Academic Press.
    1. Gray EG
    (1959)
    Axo-somatic and axo-dendritic synapses of the cerebral cortex: an electron microscope study
    Journal of Anatomy 93:420–433.
    1. Waters J
    2. Larkum M
    3. Sakmann B
    4. Helmchen F
    (2003)
    Supralinear Ca2+ influx into dendritic tufts of layer 2/3 neocortical pyramidal neurons in vitro and in vivo
    The Journal of Neuroscience 23:8558–8567.

Decision letter

  1. Carol A Mason
    Reviewing Editor; Columbia University, United States
  2. Eve Marder
    Senior Editor; Brandeis University, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

This study uses 3-D electron microscopy to map the inhibitory and excitatory synaptic input distribution of the apical dendrites of pyramidal cells in the mouse cortex. The authors compare excitatory/inhibitory synapse density in L2, L3 and L5 pyramidal cells in different cortical regions, at the bifurcation point from the apical dendritic trunk and the distal dendritic tufts. The resultant connectomic principles for the inputs onto pyramidal cells at their apical dendrites support specific computational properties of L2, L3 and subtypes of L5 pyramidal cells, and is a rich data set that should be of wide interest in the field.

Decision letter after peer review:

Thank you for submitting your article "Cell-type specific innervation of cortical pyramidal cells at their apical tufts" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Eve Marder as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

The reviewers were in the main positive about your study mapping the inhibitory and excitatory synaptic input distribution of the apical dendrites of pyramidal cells in the mouse cerebral cortex. Your comparison of excitatory/inhibitory synapse density in L2, L3 and L5 pyramidal cells in different cortical regions, at the bifurcation point from the apical dendritic trunk and the distal dendritic tufts, provides a very rich data set, one that should be of great interest to the field.

The reviewers found your results novel for the level of anatomical analysis and for implications of differential modes of inhibition for pyramidal cells in the cortex, including the following rules of synapse distribution along the apical dendrite: a. At the bifurcation point at the L1/2 border, inhibitory synapse density is higher in L2 cells than in deeper layer cells; b. At the distal apical tuft, the excitatory synapse density is lower in L5 cells than in L2 cells; c. Dendrites at the L1/2 border of L2 and deeper layer pyramidal cells are innervated by axons of different inhibitory cells. These results suggest that the computational properties of apical dendrites are distinct in L2 and L5 pyramidal cells.

Essential revisions:

Even with this praise, the reviewers had issues with the definition of the structures you analyzed, specifically, the criteria for classification of E and I synapses, for parsing orientation of distal dendrites, and for identifying axonal inputs. Many of these amendments can be textual, yet the first of these major points in particular, may affect your analysis.

1) Classification of synapses: Two of the reviewers at first assumed that you had classified synapses by the classical definition, i.e. symmetric as inhibitory and asymmetric as excitatory, but you state that "Shaft synapses, synapses on spine necks and secondary spine innervations were treated as inhibitory. Primary spine innervations were treated as excitatory synapses (Figure 1, 3)". a) Whereas one could conclude from the literature, that a single spine innervation represents an excitatory synapse, and a doubly innervated spine having one inhibitory and one excitatory input (although dual excitatory innervations have been observed previously), the reasoning goes astray for shaft synapses, which have been shown to represent both, with recent studies indicating that 15-20% of asymmetric synapses (presumably glutamatergic) are made on the dendritic shaft in the upper layers of cortex.

b) Figure 2E, F, G, indicates that some of axons innervating a shaft then also make a synapse on a spine without another input more frequently than on a spine with another input. Most spines with an inhibitory input usually have another excitatory input in the neocortex. The axons making a single synapse on spines may be thus be excitatory; please explain.

c) Validation of your classification system is requested: Discuss the differences between your assumptions and the literature, and the possible impact of your assumptions on the presented connectomic analysis. Do you or others have evidence that shaft synapses are gephyrin-positive, for example, beyond the Lascone study you cite (in BioRxiv)?

2) Identification of apical dendrites: a) You based this identification on radial direction and diameter. Whereas this may apply for deep dendrites, there might be some ambiguity for superficial cells. How exactly are L2 pyramids distinguished from other cell types in superficial layers? Can you provide quantitative characteristics of the dendrites analyzed, and possibly of some dendrites and their somata that were not considered to belong to pyramids? Such information would provide a baseline for future studies, and would avoid generalization and possible biases.

b) You assumed that dendritic branches from thicker dendrites around the L1/2 border represent bifurcation points from an apical dendritic trunk. L2 pyramidal cells were reconstructed down to their somata, but deeper layer pyramidal cells not. L5 thick-tufted pyramidal cells frequently have the bifurcation points in the center of L2/3. Is it possible that only L5 thin-tufted cells are analyzed in this study. This sampling bias may affect the conclusions.

c) The authors make no distinction between thick tufted L5 (possibly L5B) and slender layer 5 (possibly L5A). There are distinct functional differences between them. It would be interesting if the authors can distinguish them and quantify the proportion of inhibitory inputs onto these two types?

3) Distinction between L2 and L3 pyramidal neurons:

A classification of pyramidal cells based on the depth of the soma may not be reliable (e.g. L2 vs. L3). Both subtypes are possibly intermingled in the more superficial regions. Another concern might be that L2 cells without a main bifurcation are excluded according to the listed criteria, as well as branches of either type that run more obliquely or even parallel to the surface. Please comment on this, and provide more numeric data such as cell body position, size, possibly branch numbers, etc. for future reference, again, to avoid over-generalization of the difference between L2 and L3.

4) Contact by axons: a) For the axons that were reconstructed, were multiple contacts with the same dendrite or perhaps even the same neuron excluded? Did this occur?

b) When discussing the axon identity in the Results section, the authors report only the axons that targeted dendritic shafts or spines, and not axons that targeted neither. In Figure 1H and Supplementary Figure 1B, the question of whether all reported axons target contact at least one spine or shaft is important in order to better understand the results.

5) Synapse density: a) You report excitatory and inhibitory densities as a function of distance, and do not report the density per unit area, which is more informative.

b) The inhibitory synapse density around the border of L1/2 is different between L2 and L5 cells. The dendritic size around the border may be different between L2 and L5 cells. It would therefore be informative to show the relation between dendrite thickness and inhibitory synapse density of the dendrites at this border.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Cell-type specific innervation of cortical pyramidal cells at their apical dendrites" for further consideration by eLife. Your revised article has been evaluated by Eve Marder as the Senior Editor, a Reviewing Editor and two peer reviewers.

Both reviewers appreciated the revisions on your study mapping the inhibitory and excitatory synaptic input distribution of the apical dendrites of pyramidal cells in the mouse cortex. Your comparison of excitatory/inhibitory synapse density in L2, L3 and L5 pyramidal cells in different cortical regions, at the bifurcation point from the apical dendritic trunk and the distal dendritic tufts, is a rich data set that should be of wide interest in the field. The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

The reviewers still have a lingering concern: While they are receptive to the concept that if an axon maintains shaft synapses in more than 90% of its dendritic contacts, it likely represents an inhibitory axon, they and I suggest that you state this at the beginning of the Results section and that you did not use the classic symmetry in the pre- and postsynaptic densities to identify inhibitory synapses. The use of the term inhibitory synapses comes before you test this with seeding method, which makes the argument a bit circular. We suggest that you start by referring to shaft and spine synapses (under Results, subsection “Inhibitory to excitatory synapse ratio”), and refer to inhibitory synapses only once you have made their verification argument (subsection “Shaft vs. spine synapses”). In the latter section you can then present a plot that contains the corrected densities or fractions. Alternatively, you could first develop the argument of Figure 2A, B, and then present the data that are currently in Figure 1, maintaining the term inhibitory – though we would prefer 'putative inhibitory' over 'inhibitory'.

In the Discussion, you could also caution that identification of excitatory and inhibitory synapses using the classical asymmetrical or symmetrical criteria was difficult because the image quality of SBEM is slightly sub-par. New Figure 3 clearly indicates that some of axons innervating a shaft next make a synapse on a spine with (not without) another input more frequently than on a spine without another input. From the revised Figures 2-4, the readers can now easily understand the logic by which to differentiate the excitatory and inhibitory axons, not just based on the single postsynaptic structure, but on consideration of successive targets. Restating these points in the Discussion would strengthen your arguments.

On a positive note, reviewer 2 wondered whether the apical dendrites analyzed here belong to thick or slender tufted pyramidal cells in layer 5 because the two types of pyramidal cells significantly differ in function. Your new analysis has revealed their differences in spine density and this reviewer applauded this finding, and also found your quantitative analysis and conclusion that dendritic size is a key factor in determining inhibitory synapse density a strong contribution.

https://doi.org/10.7554/eLife.46876.sa1

Author response

Essential revisions:

[…] The reviewers had issues with the definition of the structures you analyzed, specifically, the criteria for classification of E and I synapses, for parsing orientation of distal dendrites, and for identifying axonal inputs. Many of these amendments can be textual, yet the first of these major points in particular, may affect your analysis.

1) Classification of synapses: Two of the reviewers at first assumed that you had classified synapses by the classical definition, i.e. symmetric as inhibitory and asymmetric as excitatory, but you state that "Shaft synapses, synapses on spine necks and secondary spine innervations were treated as inhibitory. Primary spine innervations were treated as excitatory synapses (Figure 1, 3)". a) Whereas one could conclude from the literature, that a single spine innervation represents an excitatory synapse, and a doubly innervated spine having one inhibitory and one excitatory input (although dual excitatory innervations have been observed previously), the reasoning goes astray for shaft synapses, which have been shown to represent both, with recent studies indicating that 15-20% of asymmetric synapses (presumably glutamatergic) are made on the dendritic shaft in the upper layers of cortex.

We fully agree with the point raised by the reviewers that a single shaft or spine synapse can in principle be excitatory or inhibitory. 3D EM data, however, allows us to reconstruct the axons presynaptic at these synapses; which provides the targets of all other of the output synapses of these axons. Therefore, by reconstructing the presynaptic axons, we can quantify in how many cases a shaft synapse is made by an axon that in fact prefers spine innervations, and vice versa. We used the reconstructions of 430 presynaptic axons from synapses of the apical dendrites in layers 1 and 2 (new Figure 2A-C), and determined their other synaptic targets. The distribution of the fraction of single-innervated spine targets of these axons were almost binary (new Figure 2B) with axons preferring either shaft or spines of dendrites with the exception of axons innervating the shafts of L5 slender tufted ADs (new Figure 2B). Quantitatively, 91 of 92 axons seeded from the shafts of L2 ADs made at least 80% of their other output synapses onto shafts, as well (and 89 of 92 axons made at least 90% of their other synapses onto shafts). Similarly, 34 of 35 axons seeded from spines at L2 ADs made at least 80% of their other synapses as primary spine innervations. Numbers for synapses seeded at DL ADs were comparable.

We then calculated a corrected inhibitory fraction based on this connectivity criteria for classifying excitatory and inhibitory input and found it to be rather unchanged for most cell types (new Figure 2C). For L5 slender tufted ADs, synapse densities were corrected for the synapse identity (blue, new Figure 5E, Figure 7B grey crosses indicate paired uncorrected values) due to the substantial spine-preferring input onto their AD shaft (44-60%).

In summary, while for single synapses, the identification of spine vs. shaft targets with the synaptic classification of excitatory vs. inhibitory is error prone, the classification of axonal type by preference of their other local output synapses yields misclassification rates of less than 7% (for similar results in layer 4, see also (Motta et al., 2019, Motta et al., 2019); it appears that the axons innervating ADs as studied here show a stronger bimodal preference for either shafts or spines than those reconstructed in L4, (Motta et al., 2019)).

b) Figure 2E, F, G, indicates that some of axons innervating a shaft then also make a synapse on a spine without another input more frequently than on a spine with another input. Most spines with an inhibitory input usually have another excitatory input in the neocortex. The axons making a single synapse on spines may be thus be excitatory; please explain.

This was an unfortunate error in former Figure 2. The labels have been corrected in current Figure 3D, 4B-C (previously as Figure 2E, F, G, respectively). The shaft-preferring axons (inhibitory) innervate mainly spines with an additional (presumably excitatory) synapse (91.35% of 821 spine synapses). They only innervate a spine without a partner 8.65% of the time. See also the additional analyses in Figure 2A-C described above.

c) Validation of your classification system is requested: Discuss the differences between your assumptions and the literature, and the possible impact of your assumptions on the presented connectomic analysis. Do you or others have evidence that shaft synapses are gephyrin-positive, for example, beyond the Lascone study you cite (in BioRxiv)?

Our connectivity metric for the identification of axonal input type suggests a very strong shaft- vs. spine- preference for axons innervating most pyramidal neurons (new Figure 2, section a above). L5st neurons, however, did receive a significant amount of input from spine-preferring axons onto their shafts. We, therefore, corrected our synapse density measurement for this cell-type specific difference. We also reviewed a few studies which investigated the fraction of asymmetric and symmetric synapses (assumed to be excitatory and inhibitory, respectively) on shaft and spine of pyramidal neurons using 3D-EM:

1) (Chen et al., 2012) used a combination of ssTEM with Teal-Gephyrin 2P imaging. The authors found 26/26 asymmetric synapses on spines (no shaft asymmetric innervation) and 6/10 inhibitory synapses on shaft of the L2/3 pyramidal neuron dendrite (length: 30 µm). 3/10 inhibitory synapses were located on double-innervated spines and 1/10 single-innervated spine.

2) (White and Rock, 1980) studied the basal dendrites of the spiny stellate neurons in L4 of primary somatosensory cortex using serial section TEM (ssTEM). They found 100% of shaft and 3% (14/359) of spine synapses to be symmetric.

3) (White and Hersch, 1982) analyzed multiple segments of apical dendrites in L5/6 pyramidal neurons using ssTEM. They found 20% of shaft synapses to be asymmetric and only 0.6% of spine synapses to be symmetric (Table 1 in (White and Hersch, 1982)).

In conclusion, the degree to which the spine/shaft preference proxies for identification of excitatory and inhibitory synapses is dendrite type specific and shall be studied in the specific region of study either using an estimation method as described here or dense reconstruction of axons where most axon could be identified based on their single-innervated spine innervation fraction.

Notably, studies where dendrite types are not considered and a volume average of asymmetric and symmetric synapses is calculated could result in high degrees of symmetric input to shafts (up to 50%, See (Kwon, MerchánPérez et al., 2018, Santuy, Rodriguez et al., 2018)).

2) Identification of apical dendrites: a) You based this identification on radial direction and diameter. Whereas this may apply for deep dendrites, there might be some ambiguity for superficial cells. How exactly are L2 pyramids distinguished from other cell types in superficial layers? Can you provide quantitative characteristics of the dendrites analyzed, and possibly of some dendrites and their somata that were not considered to belong to pyramids? Such information would provide a baseline for future studies, and would avoid generalization and possible biases.

To disambiguate the morphology of the apical dendrites used in our analysis, we reconstructed the shaft morphology of all pyramidal neurons used in our cell-type analysis (new Figure 5, Figure 7, new Figure 5—figure supplement 1 contains a cell type gallery of all reconstructed ADs from LPtA and PPC-2 datasets). We also added diameter measurements (new Figure 2F). In addition, we specifically looked for pyramidal neurons with obliquely directed apical dendrites (marginal neurons) and measured their inhibitory input and compared it to other L2 pyramidal neurons (new Figure 5, Figure 5—figure supplement 1). In the two examples we found of L2 marginal pyramidal neurons, we did not find evidence that their input fraction was different from L2 pyramidal cells. For the distinction of L2 and L3 pyramidal cells, we again were able to use our larger EM datasets for clear differentiation (see cell type gallery in Figure 5—figure supplement 1).

b) You assumed that dendritic branches from thicker dendrites around the L1/2 border represent bifurcation points from an apical dendritic trunk. L2 pyramidal cells were reconstructed down to their somata, but deeper layer pyramidal cells not. L5 thick-tufted pyramidal cells frequently have the bifurcation points in the center of L2/3. Is it possible that only L5 thin-tufted cells are analyzed in this study. This sampling bias may affect the conclusions.

We reconstructed all the pyramidal neurons used in our cell-type analysis in the high- and low-resolution segments of LPtA and PPC-2 datasets down to their soma of origin for L2-5 neurons (new Figure 5—figure supplement 1). We found the AD morphology of the former L5 group much closer to L5B (thick-tufted, 7/10) neurons. Next, we identified six new L5 neurons with smaller soma and slender dendrite morphology (Larkman and Mason, 1990) (L5 slender tufted neurons or L5A, new Figure 5A, new Figure 5—figure supplement 1A). Next, we used hierarchical clustering to classify L5 neurons based on their soma diameter, AD trunk diameter, number of oblique dendrites and main bifurcation depth (new Figure 5B-C). In addition, these neurons had a substantially lower spine density (new Figure 5D). L5st neurons also seemed to have an increased inhibitory input fraction (15-31%) as compared to L5tt neurons in upper cortex (new Figure 7C).

c) The authors make no distinction between thick tufted L5 (possibly L5B) and slender layer 5 (possibly L5A). There are distinct functional differences between them. It would be interesting if the authors can distinguish them and quantify the proportion of inhibitory inputs onto these two types?

We thank the reviewers for the insightful suggestion – the additional analyses of

L5 slender tufted cells yielded an interesting specialization in input fraction (new Figure 5, Figure 7).

3) Distinction between L2 and L3 pyramidal neurons:

A classification of pyramidal cells based on the depth of the soma may not be reliable (e.g. L2 vs. L3). Both subtypes are possibly intermingled in the more superficial regions. Another concern might be that L2 cells without a main bifurcation are excluded according to the listed criteria, as well as branches of either type that run more obliquely or even parallel to the surface. Please comment on this, and provide more numeric data such as cell body position, size, possibly branch numbers, etc. for future reference, again, to avoid over-generalization of the difference between L2 and L3.

We explicitly looked for L2 pyramidal neurons with an apical dendrite running oblique to the pial surface and found two of these marginal neurons (L2MN, new Figure 5, Figure 5—figure supplement 1A). We also compared their synaptic input composition with other L2 neurons (Results section, new Figure 5E, Figure 6A, Figure 7B). Our preliminary analysis (n=2) fails to rule out the possibility that L2MN neurons have a similar inhibitory fraction to other L2 neurons. For the distinction between L2 and L3, please see the new cell type gallery. Also, we report the relation between synaptic input composition and cell body depth, and AD length (Figure 6), which should avoid any overly binary distinctions.

4) Contact by axons: a) For the axons that were reconstructed, were multiple contacts with the same dendrite or perhaps even the same neuron excluded? Did this occur?

We analyzed the frequency of multiple contacts with ADs (new Figure 3E-G) and its effect on our conclusions. Multiple contacts within the EM volumes we analyzed occurred in 23% of the cases, such that the preference of axons for innervating certain types of ADs remained rather substantial even when disregarding the multiple innervations (Figure 3G).

b) When discussing the axon identity in the Results section, the authors report only the axons that targeted dendritic shafts or spines, and not axons that targeted neither. In Figure 1H and Supplementary Figure 1B, the question of whether all reported axons target contact at least one spine or shaft is important in order to better understand the results.

All axons reported in the manuscript were seeded from either spine or shaft synapses of an AD. Thus, they innervated at least the shaft or spine of the seed dendrite. For their remaining targets, (example: new Figure 2A), we identified these for a subset of axons, and report the distribution of axonal targets in Figure 3D and Figure 4 – but always for axons that targeted at least one spine or one shaft of an AD. We added a statement to that end to the Results subsection “Comparative analysis of innervation preference between cortex types”.

5) Synapse density: a) You report excitatory and inhibitory densities as a function of distance, and do not report the density per unit area, which is more informative.

We thank the reviewers for this suggestion. We additionally measured the diameter of apical dendrites and used it to calculate the synapse density per unit surface area of each cell type (new Figure 2F-G). All conclusions remained unaltered when normalizing for surface area of apical dendrites instead of their shaft path length.

b) The inhibitory synapse density around the border of L1/2 is different between L2 and L5 cells. The dendritic size around the border may be different between L2 and L5 cells. It would therefore be informative to show the relation between dendrite thickness and inhibitory synapse density of the dendrites at this border.

We added the relationship between the apical dendrite diameter and inhibitory/excitatory synapse density for apical dendrites of different cell types (new Figure 2—figure supplement 1). We found a correlation between inhibitory synapse density and the diameter of the apical dendrite. However, the diameter differences were not substantial enough to change the patterns of synapse density observed using the distance-based metric (new Figure 2G).

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

[…] The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

The reviewers still have a lingering concern: While they are receptive to the concept that if an axon maintains shaft synapses in more than 90% of its dendritic contacts, it likely represents an inhibitory axon, they and I suggest that you state this at the beginning of the Results section and that you did not use the classic symmetry in the pre- and postsynaptic densities to identify inhibitory synapses. The use of the term inhibitory synapses comes before you test this with seeding method, which makes the argument a bit circular. We suggest that you start by referring to shaft and spine synapses (under Results, subsection “Inhibitory to excitatory synapse ratio”), and refer to inhibitory synapses only once you have made their verification argument (subsection “Shaft vs. spine synapses”). In the latter section you can then present a plot that contains the corrected densities or fractions. Alternatively, you could first develop the argument of Figure 2A, B, and then present the data that are currently in Figure 1, maintaining the term inhibitory – though we would prefer 'putative inhibitory' over 'inhibitory'.

In the Discussion, you could also caution that identification of excitatory and inhibitory synapses using the classical asymmetrical or symmetrical criteria was difficult because the image quality of SBEM is slightly sub-par. New Figure 3 clearly indicates that some of axons innervating a shaft next make a synapse on a spine with (not without) another input more frequently than on a spine without another input. From the revised Figures 2-4, the readers can now easily understand the logic by which to differentiate the excitatory and inhibitory axons, not just based on the single postsynaptic structure, but on consideration of successive targets. Restating these points in the Discussion would strengthen your arguments.

On a positive note, reviewer 2 wondered whether the apical dendrites analyzed here belong to thick or slender tufted pyramidal cells in layer 5 because the two types of pyramidal cells significantly differ in function. Your new analysis has revealed their differences in spine density and this reviewer applauded this finding, and also found your quantitative analysis and conclusion that dendritic size is a key factor in determining inhibitory synapse density a strong contribution.

We appreciate your comments and have made all changes to the description of synapses as requested:

- We start by describing shaft and spine synapses, only (subsection “Putative inhibitory to excitatory synapse ratio”);

- Switch to the term putative inhibitory after the calibration (subsection “Shaft vs. spine synapses” ff);

- Added a cautionary statement to the beginning of the Results;

- Added a paragraph to the Discussion (subsection “Distinction of excitatory vs. inhibitory synapses”).

https://doi.org/10.7554/eLife.46876.sa2

Article and author information

Author details

  1. Ali Karimi

    Department of Connectomics, Max Planck Institute for Brain Research, Frankfurt, Germany
    Contribution
    Resources, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology
    Contributed equally with
    Jan Odenthal
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6477-2523
  2. Jan Odenthal

    Department of Connectomics, Max Planck Institute for Brain Research, Frankfurt, Germany
    Contribution
    Data curation, Formal analysis, Validation, Visualization
    Contributed equally with
    Ali Karimi
    Competing interests
    No competing interests declared
  3. Florian Drawitsch

    Department of Connectomics, Max Planck Institute for Brain Research, Frankfurt, Germany
    Contribution
    Resources, Data curation, Validation, Investigation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9543-1417
  4. Kevin M Boergens

    Department of Connectomics, Max Planck Institute for Brain Research, Frankfurt, Germany
    Contribution
    Data curation, Investigation, Methodology
    Competing interests
    No competing interests declared
  5. Moritz Helmstaedter

    Department of Connectomics, Max Planck Institute for Brain Research, Frankfurt, Germany
    Contribution
    Conceptualization, Formal analysis, Supervision, Validation, Visualization
    For correspondence
    mh@brain.mpg.de
    Competing interests
    Reviewing editor, eLife
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7973-0767

Funding

Max-Planck-Gesellschaft (Open-access funding)

  • Ali Karimi
  • Jan Odenthal
  • Florian Drawitsch
  • Kevin M Boergens
  • Moritz Helmstaedter

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Matthew Larkum, Johannes Letzkus and Idan Segev for discussions, Benedikt Staffler, Emmanuel Klinger, Manuel Berning, Alessandro Motta for computational and Jakob Straehle, Meike Schurr, Yunfeng Hua for experimental advice, and Alessandro Motta and Benedikt Staffler for contributing code. We thank M S E A Aly, L Bezzenberger, A B Brandt, B Heftrich, A C Rix, B L Stiehl, C Arras, C M Schumm, D E Celik, D J Goffitzer, J Buß, K M Trares, K Weber, L Buxmann, L Decker, L C R Kreppner, M S Kronawitter, N M Böffinger, N Plath, S M Bohne, S Reichel, T Engelmann, T Ernst, T Winkelmeier, V C Kalbert, K Kramer, L Präve, M Präve, N Berghaus, O J Brandt, S S Wehrheim for neurite reconstructions, Heiko Wissler, Susanne Babl, Lisa Bezzenberger, Alexander Brandt, Raphael Jakoby, Raphael Kneißl and Marc Kronawitter for annotator training and task management and Heiko Wissler for support with visualizations.

Ethics

Animal experimentation: All experimental procedures were performed according to the law of animal experimentation issued by the German Federal Government under the supervision of local ethics committees and according to the guidelines of the Max Planck Society. The experimental procedures were approved by Regierungspräsidium Darmstadt, under protocol ID V54 - 19c20/15 F126/1015 (LPtA, PPC-2) or V54 - 19 c 20/15 - F126/1002 (V2, PPC, ACC). The S1 sample was prepared following experimental procedures approved by Regierung von Oberbayern, 55.2-1-54-2532.3-103-12.

Senior Editor

  1. Eve Marder, Brandeis University, United States

Reviewing Editor

  1. Carol A Mason, Columbia University, United States

Publication history

  1. Received: March 14, 2019
  2. Accepted: February 26, 2020
  3. Accepted Manuscript published: February 28, 2020 (version 1)
  4. Version of Record published: June 16, 2020 (version 2)

Copyright

© 2020, Karimi et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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