Figures and data
L1 and L2 in the human TLN as revealed in methylene-blue stained semithin sections, Golgi-impregnation and low power EM.
A, Methylene-blue stained semithin section of L1a, L1b, and L2. The cell sparse zone contains degenerating neurons (dark appearance, arrowheads in the framed area) and a cell body (asterisk in the framed area). Note the sudden increase in the density of neurons at the L1b/L2 border. Scale bar 500 µm. B, C, Golgi-impregnated radial astrocytes in L1a (B) and L1b (C). Scale bars 50 µm. D, Group of neurons in L1b containing lipofuscin granules of various shape and size (asterisks) most prominent in two of the neurons. Scale bar 50 µm. E, Putative CR-cell identifiable by its long horizontally oriented dendrite (marked by asterisks) and an inverted putative GABAergic interneuron (asterisk) in L1b. Scale bar 50 µm. F, G, Golgi-impregnated CR-Cells in L1a with the characteristic horizontal orientation of dendrites (arrowheads) and axons (asterisks). Scale bars 50 µm. H, EM micrograph of L1a (astrocytic layer, astl) underneath the pial surface. Note the abrupt transition (indicated by the dashed line) to a neuropil (neurl) containing two putative CR-cells (asterisks), a reactive microglial cell (arrowhead), and dendrites and synapses. Scale bar 25 µm. I, EM micrograph of L1b containing thousands of dendritic profiles, a putative GABAergic interneuron (framed area, asterisk) but also degenerating neurons (framed area, arrowheads) and a reactive microglia (I1) Scale bar 50 µm.
Structural and synaptic organization of L1 in the human TLN as revealed by EM.
A, Superficial part of L1a composed of a dense network of fine astrocytic processes (asterisks). In the reactive microglial cell (rmic) so-called lipofuscin granules (framed area) were observed. Below the astrocytic network synaptic complexes gradually increased (highlighted in blue for target structures, yellow for SBs). Scale bar 10 µm. B, Two apical dendrites (blue) traversing L2 with beginning terminal tuft dendrites in L2 and L1 contacted by SBs (transparent yellow); some of which are putatively GABAergic terminating on the dendritic shaft (asterisks). Scale bar 2.5 µm. C, Large GABAergic SB (sb, transparent yellow) identifiable by the small-sized ovoid SVs and the less prominent PSD (arrowheads) synapsing on a dendritic shaft (de, transparent blue) in L1b. D, Three SBs in L1a (sb1-sb3, transparent yellow) located on a dendritic spine (sp, transparent blue) and a neighboring dendritic shaft (sh, transparent blue). Note that sb1 is excitatory as indicated by the larger round SVs with a prominent AZ whereas sb2 and sb3 are putative inhibitory terminals identified by smaller ovoid SVs and the lack of a prominent PSD. E, Elongated spine (sp) emerging from a small caliber dendrite (de, transparent blue) receiving input from a small-sized SB (sb, transparent yellow). Note the large, elongated spine apparatus (framed area). Scale bar in C-E 0.5 µm. In graphs C-E AZs are marked by arrowheads.
3D-volume reconstructions of SBs and vesicle pools in L1a and L1b of the human TLN. A-D,
Here, all SBs are colored in transparent yellow. Subelements: PreAZs (red), SVs (green), DCVs (magenta), mitochondria (white). The postsynaptic target structures are given in blue A, Six SBs (sb1-sb6) terminating at different locations on a dendritic segment in L1b, two of which (syn3 and syn4) were located on spines, the remaining on the dendritic shaft. Scale bar 1 μm. B, Large SB terminating on two dendritic segments of different shape and size (de1, de2) in L1a. C, SB synapsing on a dendrite (de) and spine (sp) in L1b containing several mitochondria associated with the pool of SVs. D, E, 3D-volume reconstructions of the total pools of SVs (green dots). Note the different shape and size of the PreAZs (red) with either a non-perforated macular (D) or perforated (E) appearance and the comparably large total pool size. Note the large DCVs intermingled with the pool of SVs.
Comparative quantitative analysis of various structural and synaptic parameters in L1 of the human TLN.
Correlation plots for structural and synaptic parameters characterizing L1 SBs.
Correlation plots showing the strength of correlations between structural and synaptic parameters (Source Data 2). Correlations dots and regression lines for L1a are given in black and that for L1b in red. A, Surface area of SBs vs. surface area of PreAZ; B, Volume of SBs vs. volume of mitochondria; C, Surface area of PreAZs vs. total pool of SVs; D, Surface area of PreAZs vs. p10 nm RRP; E, Surface area of PreAZs vs. p20 nm RRP; F, Surface area of PreAZs vs. p60-p200 nm RP; G, Surface area of PreAZs vs. p200 nm resting pool; H, Surface area of PreAZs vs. p500 nm resting pool.
EM tomography of SBs in L1a and L1b in the human TLN.
A, Large SB (sb) terminating with a single AZ (arrowheads) on a terminal tuft dendritic shaft (de) in L1a. B, SB (sb) synapsing on a stubby spine (sp) with a prominent spine apparatus (framed area) and a thick dendritic segment (de) in L1b. Scale bars 1 µm. C, Large mushroom (sp1) and a small spine head (sp2) receiving input from three SBs (sb1, sb2, sb3) and a single SB (sb4) in L1b. Note the large AZs (arrowheads) and the prominent spine apparatus (red framed area) in the mushroom spine. Scale bar 0.5 µm. D, E, F, High-power images of ‘docked’ SVs (arrowheads) taken from a tilt-series through an individual PreAZ at a L1a spine SB. Note the so-called omega-shaped bodies in (E, F) pointing to the already opening and release of glutamate quanta. Scale bars 0.1 µm.
’Docked SVs’ in L1a and L1b of the human TLN.
Astrocytic coverage of synaptic complexes in the human TLN.
A, EM micrograph of two adjacent SBs (sb1, sb2) terminating on two spines (sp1, sp2) in L1a. Sp2 contained a prominent spine apparatus (framed area), sb1 two DCVs (asterisks). Note that both synaptic complexes were only partially ensheathed by fine astrocytic processes (transparent green) reaching the AZs only on one side. In both synaptic complexes two ‘docked’ SVs (transparent red) at the PreAZs were found. Scale 0.25 µm. B, Stubby spine (stsp) receiving two SBs (sb1, sb2) in L1b. Here, fine astrocytic processes (transparent green) were found close to the two synaptic complexes but never reached the synaptic cleft. Note also the ‘docked’ SVs (transparent red) pointing to multivesicular release. In both images the AZs are marked by arrowheads. Scale bar 0.25 µm. C, Bar histogram showing the percentage (mean ± SD) of the volumetric fraction of astrocytic processes to the total volume in L1 to L6. Values for L6 are taken from Schmuhl-Giesen et al. 2022. The horizontal bars indicate significant differences between cortical layers *: p < 0.05 (Source Data 4).
Schematic drawing of the possible wiring of L1 in the human TLN.
The possible neuronal and synaptic organization of neocortical L1 in humans modified and based on findings from rat neocortex (reviewed by Schuman et al. 2021). The dendritic domain of excitatory neurons is given in blue, the axonal projection and connectivity in magenta and that of different GABAergic interneurons in red and brown, respectively. Furthermore, a subpopulation of persistent CR-cells is illustrated in the diagram.
Abbreviations: α7: nicotinic receptor-expressing interneuron; CanC: canopy cell; ChC: chandelier cell; L: layer; NGFC: neurogliaform cell; PC: pyramidal cell; SST: somatostatin containing interneuron; VIP: vasoactive intestinal peptide-expressing interneuron; VIP/Bip: vasoactive intestinal peptide–expressing bipolar interneuron; VIP/CCK: cholecystokinin- and vasoactive intestinal peptide-expressing interneuron.
Box plots of various structural parameters in L1 of the human TLN
Data distributions for each patient are indicated by the medians (horizontal bars), IQRs (framed areas), minimum and maximum (vertical lines) for the distribution of: A, Surface area of SBs; B, Volume of SBs; C, Surface area of PreAZs; D, Surface area of PSD; E, Volume of mitochondria. Significant differences between different patients are indicated by asterisks. Note that several structural parameters are significantly different. The significant bars highlighted in red indicate significant differences between sublaminae L1a and L1b in the same patient. *: p < 0.05; **: p < 0.01; ***: p < 0.001 (Source Data 2).
Box plots of various synaptic parameters in L1 of the human TLN
Data distributions for each patient are indicated by the medians (horizontal bars), IQRs (framed areas), minimum and maximum (vertical lines) for the distribution of: A, Total pool of SVs; B, SVs in the p10 nm RRP; C, SVs in the p20 nm RRP; D, SVs in the p6-200 nm RP; E, SVs in the > p200 nm resting pool; Note that several structural parameters are not significantly different. The significant bars highlighted in red indicate significant differences between sublaminae L1a and L1b in the same patient. *: p < 0.05; **: p < 0.01; ***: p < 0.001 (Source Data 2).
