Article Figures & Data
Figures
- Figure 1.
Distribution of inhibitory GABA myelinated axons in human temporal cortex. A, A 70-nm-thick section through human cortex immunostained with MBP (white) and GABA (red). Nuclei are labeled with DAPI (blue). B, Volume reconstruction of a subregion from layer 3a (35 serial sections, 70 nm each). Several GABA myelinated axons are marked with yellow asterisks. C, Proportion of myelinated axons that contain GABA in human temporal cortex. Means and SEs from eight human patients are shown. D, Density of nonGABA and GABA myelinated axonal profiles. Means and SEs from eight human patients are shown. E, A 70-nm-thick section through mouse cortex (MBP, white; GABA, red; and DAPI, blue) shown at the same scale as the human cortical section in panel A. Note the difference in cortical thickness between the two species. F, Comparison of the proportion of GABA myelinated axons and the density of myelinated axons throughout the layers of human and mouse cortex (means and SEs from eight human patients and six mice are shown). For this comparison, human results for layers 2, 3a, and 3b were pooled together to compare with mouse cortical layer 2/3. *p < 0.05, **p ≤ 0.01.
- Figure 2.
Myelinated GABA axons have distinct cytoskeletal composition and shorter nodes of Ranvier, compared to nonGABA myelinated axons. A, Volume reconstruction of 35 serial sections (70 nm each) from layer 3a of human cortex immunolabeled with GABA (red), MBP (white), neurofilament heavy chain (green), and α-tubulin (cyan). A single section from the boxed region is shown in C. B, Analysis of the cytoskeletal content of myelinated GABA versus nonGABA axons from layers 3a, 3b, 4, and 5 from three human samples (216 nonGABA and 164 GABA myelinated axons). C, A single section from the boxed region in A, showing different combinations of immunostains. Note that myelinated GABA axons are brightly labeled with the neurofilament antibody but have weak tubulin immunoreactivity. D, Maximum projection from three serial sections showing a node of Ranvier (yellow arrowheads) from a nonGABA axon. At the node, which is devoid of MBP immunofluorescence, the axon can be followed using the tubulin immunofluorescence (cyan). E, Node of Ranvier (yellow arrowheads) from a GABA axon. F, Comparison of the lengths of the nodes of Ranvier from GABA and nonGABA cortical axons (39 GABA and 100 nonGABA nodes, eight samples).
- Figure 3.
The majority of GABA myelinated axons are parvalbumin-immunopositive. A, Two consecutive sections from layer 3b of human cortex, immunolabeled for GABA (red), parvalbumin (green), and MBP (white). Cell nuclei are stained with DAPI (blue). Four myelinated GABA axonal profiles are marked with numbers; three of them (1, 2, and 4) are immunoreactive for parvalbumin. B, Pie charts showing the distribution of GABA and nonGABA myelinated profiles according to their parvalbumin immunoreactivity. Parvalbumin immunofluorescence was normalized for each sample (186 GABA and 172 nonGABA axonal profiles from two different samples). C, PV neuron and processes in layer 3b of human cortex from a biopsy; volume reconstruction from 49 serial sections. Yellow arrowheads point to PV-positive myelinated axons. D, Cytoskeletal composition and node length of myelinated axons in biopsy tissue. Left, Comparison of the immunofluorescence intensity (mean ± SE) for neurofilament heavy chain and α–tubulin within myelin profiles of nonPV (blue) and PV axons (green) from the biopsy sample. The differences are statistically significant (p < 0.001 for NFH and p < 0.0001 for α-tubulin; 48 nonPV and 26 PV axons from layers 3a, 3b, and 4 from one sample). Right, Nodes of PV axons are shorter than nonPV axons in the biopsy sample (p < 0.01; 34 nonPV and 24 PV nodes). E, Cytoskeletal composition and node length of myelinated axons in epilepsy surgery tissue. Left, Comparison of the immunofluorescence intensity for neurofilament heavy chain and α–tubulin within myelin profiles of nonGABA (blue) and GABA axons (red) from epilepsy surgery samples. The differences are statistically significant (p < 0.0001, 196 nonGABA and 177 GABA axons from layers 3a, 3b, and 4 from three different samples). Right, Nodes of GABA axons are shorter than nonGABA axons in epilepsy surgery samples (p < 0.0001; 100 nonGABA and 39 GABA nodes from eight different samples). **p <0.01.
- Figure 4.
GABA myelinated axons have more mitochondria. A, A single 70-nm section from layer 4 of human cortex immunolabeled with GABA (red), PLP (white), MDH2 (magenta), TOMM20 (green), and VDAC1 (cyan). B, The boxed area in A is enlarged to show two immunolabeled mitochondria, with the MDH2 mitochondrial matrix protein surrounded by the outer mitochondrial membrane proteins TOMM20 and VDAC1. C, Five serial sections through axons 1 and 2, marked on A, top section. Axon 1 is nonGABA and axon 2 is GABA. Adjacent to axon 2, there is also a nonGABA axon. D, Boxplots of the immunofluorescence for MDH2, TOMM20, and VDAC1 within nonGABA and GABA myelinated axons. Center lines show the medians, box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th to the 75th percentiles, outliers are represented by dots, n = 88 and n = 41 axons for MDH2, n = 95 and n = 43 axons for TOMM 20, and n = 97 and n = 47 axons for VDAC1, from layers 3a through 5 from three different samples.
- Figure 5.
GABA myelinated axons have more CNP in their myelin. A, Volume reconstruction of 21 serial sections from human cortical layer 5, immunostained with CNP (magenta) and MBP. B, Three consecutive sections through a GABA myelinated axon immunolabeled for CNP (magenta) and MBP (white). The first section is also shown immunolabeled for GABA. C, Thicker GABA axons (0.6–1.2 μm in diameter) have significantly more CNP in their myelin sheath compared to nonGABA axons of similar thickness. Center lines in boxplots show the medians, box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th to the 75th percentiles, outliers are represented by dots. For the boxplot on the left (axon diameter 0.1–0.6 μm), n = 341 nonGABA and n = 98 GABA myelinated axons from two samples), and for the boxplot on the right (axon diameter 0.6–1.2 μm), n = 142 nonGABA and n = 34 GABA axons from two samples).
Tables
Patient Gender Age Clinical diagnosis Region Fixative Q1010 Male 30 Epilepsy Anterior lateral temporal cortex 2% PFA, 2% GA in PB Q1011 Male 44 Epilepsy Anterior medial temporal cortex 4% PFA, 1% GA, 2.5% DMSO in PB Q1014 Female 48 Epilepsy Anterior lateral temporal cortex 2% PFA, 4% GA, 2.5% DMSO in CB Q1015 Female 66 Epilepsy Anterior lateral temporal cortex 2% PFA, 4% GA, 2.5% DMSO in CB Q1016 Male 49 Epilepsy Lateral temporal cortex 2% PFA, 4% GA, 2.5% DMSO in CB Q1017 Male 46 Epilepsy Lateral temporal cortex 2% PFA, 4% GA, 2.5% DMSO in CB Q1018 Female 20 Epilepsy Lateral temporal cortex 2% PFA, 4% GA, 2.5% DMSO in CB Q1019 Female 51 Epilepsy Lateral temporal cortex 2% PFA, 4% GA, 2.5% DMSO in CB 10/16/14 Female 30 Nonspecific inflammation of the white matter Frontal cortex 4% PFA in PB CB, cacodylate buffer; DMSO, dimethyl sulfoxide; GA, glutaraldehyde; PB, phosphate buffer; PFA, paraformaldehyde.
Antigen Host Antibody source Dilution RRID MBP Chicken AVES MBP 1:200 RRID:AB_2313550 GABA Guinea pig Millipore AB175 1:5000 RRID:AB_91011 Parvalbumin Rabbit SWANT PV28 1:300 RRID:AB_2315235 Neurofilament heavy Chicken AVES NFH 1:100 RRID:AB_2313552 α-Tubulin Rabbit Abcam ab18251 1:100 RRID:AB_2210057 PLP Chicken AVES PLP 1:100 RRID:AB_2313560 CNPase Chicken AVES CNP 1:100 RRID:AB_2313538 MDH2 Rabbit Origene TA308153 1:200 RRID:AB_2722674 TOMM20 Rabbit Abcam ab78547 1:100 RRID:AB_2043078 VDAC1 Rabbit ProteinTech 10866-1-AP 1:50 RRID:AB_2257153 Antibody controls are presented in Extended Data Table 2-1, Figure 2-2. RRID, Research Resource Identifier; MBP, myelin basic protein; PLP, proteolipid protein; CNPase, 2’,3’-cyclic nucleotide 3’-phosphodiesterase; MDH2, malate dehydrogenase 2; TOMM20, translocase of outer mitochondrial membrane 20; VDAC1, voltage dependent anion channel 1.
Extended Data Table 2-1
Antibody controls: Pearson’s correlation (PC) coefficients from four different control experiments. The comparison between adjacent sections tests the consistency of staining, as the distribution of targets is very similar on two adjacent ultrathin sections (70-nm thickness). This correlation is influenced by antibody characteristics, but also the size of targets, with smaller targets displaying larger spatial variability from section to section, and therefore a smaller PC coefficient. The comparison with an antibody against an overlapping antigen is a test for the specificity of staining. The following comparisons were done: MBP/PLP, MBP/CNP, MDH2/TOMM20, MDH2/VDAC1. The lower PC coefficient for CNP reflects the fact that CNP is present only in certain regions of the myelin sheath, while MBP is an integral protein of myelin. The antibodies against mitochondrial proteins (MDH2, TOMM20, and VDAC1) also have lower PC coefficients, because TOMM20 and especially VDAC1 antibodies give a rather sparse punctate immunolabeling of mitochondria (Fig. 4; Extended Data Figure 2-2), and even when labeling the same mitochondrion, the immunofluorescence signal from the different antibodies often does not overlap. Another test for specificity is the comparison with an antibody against a spatially exclusive antigen. PC coefficient values of 0 and below are expected in this case. MBP, PLP, and CNP (present in the myelin sheath) were each compared with GABA (inside inhibitory neurons); MDH2, TOMM20, and VDAC1 (mitochondria) were compared with MBP (myelin sheath). And finally, all antibodies were compared with DAPI to control for background nuclear staining. Download Table 2-1, DOCX file.
Extended Data figure 2-2
Immunolabeling of mitochondria on LRWhite sections from mouse neocortex. Each image is a MAX projection from four serial sections, 70 nm each. The grey image is the tissue autofluorescence imaged in the 488 channel, and it is superimposed with the immunofluorescence from a mitochondrial marker in green (left column) or with the DAPI label of nuclei in blue (right column). Arrowheads point to several mitochondria, which can be seen as brighter elongated structures within the neuronal cell bodies. There are also abundant mitochondria within the neuropil, but they are harder to distinguish in the autofluorescence image. Download Figure 2-2, TIF file.
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