www.sciencemag.org/content/344/6181/319/suppl/DC1

Supplementary Materials for

Distinct Profiles of Myelin Distribution Along Single Axons of Pyramidal Neurons in the Neocortex

Giulio Srubek Tomassy, Daniel R. Berger, Hsu-Hsin Chen, Narayanan Kasthuri, Kenneth J. Hayworth, Alessandro Vercelli, H. Sebastian Seung, Jeff W. Lichtman, Paola Arlotta*

*Corresponding author. E-mail: paola_arlotta@harvard.edu

Published 18 April 2014, Science 344, 319 (2014) DOI: 10.1126/science.1249766

This PDF file includes

Materials and Methods Supplementary Text Figs. S1 to S5 Tables S1 and S2 References Movies S1 to S3

Other Supplementary Material for this manuscript includes the following: (available at www.sciencemag.org/content/344/6181/319/suppl/DC1)

Movies S1 to S3

Materials and Methods:

Mice Wildtype C57Bl/6 mice were purchased from Charles River Laboratories. Dab1-/- mice

were purchased from Jackson Laboratories. Emx1-Cre;RhoAfl/fl mice were previously

described(22). All animal studies were approved by the Harvard University Institutional

Animal Care and Use Committee (IACUC) and performed in accordance with

institutional and federal guidelines.

Tissue Preparation and Electron Microscopy For the generation of the adult mouse S1 dataset, brain samples were prepared as

described previously(26). Briefly, one adult mouse was perfused with 2.5%

glutaraldehyde/2.0% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.4).

Tissues were dissected, fixed for 2–4 hrs in the same fixative, rinsed, and stored at 4°C in

0.1 M cacodylate buffer (pH 7.4). The samples were processed according to the ROTO

protocol(26), dehydrated in graded ethanol solutions, and embedded in epoxy resin

(Polybed, Polysciences, Warrington) following standard procedures. The cerebral cortex

was cut and collected on kapton tape (glow discharged to prevent wrinkling of sections)

with an ATUM (automatic tape-collecting ultra microtome) at 30 nm slice thickness(27,

28)(28). The tape containing all the sections was cut into strips, mounted on 4 inch

silicon wafers (University Wafers, South Boston) and then carbon coated (Denton 502B,

Moorestown) to provide grounding for the electron imaging. Every eighth section was

imaged using a Zeiss Sigma scanning electron microscope at a resolution of 30x30 nm

per pixel (carbon-coated, backscatter imaging(29)), yielding an image stack with a voxel

size of 30x30x240 nm and a total imaged volume of ~ 1000x500x61 µm3.

Tracing and rendering

Cell bodies, axons and myelin sheaths were labeled manually throughout the EM image

stacks, using a software tool (Volume Annotation and Segmentation Tool, VAST). VAST

allows users to draw in colors over voxel data sets. All tracings can be reproduced using

the TrakEM2 plug-in of the Fiji framework(10) . Resulting labeled images and meta-data

about the labels were exported and processed externally. For rendering, 3D surface

meshes of labeled objects were generated from the exported data using MATLAB scripts

developed in house (The MathWorks Inc.) and final 3D renderings were generated using

3ds Max (Autodesk Inc.). To measure distances and lengths, fiducials were painted at

points of interest in VAST, and MATLAB scripts generated in house were used to

compute Euclidian distances between such fiducials. In the V1 data set the pia surface is

horizontal and therefore a single Y coordinate was used to compute the distance of each

label from the pia. In the S1 data set the pia is oblique and therefore three points on the

pial surface were used to approximate the position of the pia and the Y coordinate of each

label was measured as the distance from that plane.

Immunohistochemistry, in situ hybridization and histology

Mouse brains for immunohistochemistry were processed as previously described(30).

Primary antibodies and dilutions were as follows: rat anti-MBP, 1:100 (Millipore,

MAB386); mouse anti-MBP, 1:100 (Abcam, ab62631); rabbit anti-CUX1, 1:100

(SantaCruz, M-222); rat anti-CTIP2, 1:1,000 (Abcam, ab18465); goat anti-SOX10, 1:100

(Santacruz, N-20); mouse anti-APC, 1:500 (Millipore, Ab-7). Appropriate secondary

antibodies were from the Molecular Probes Alexa series and the Vectastain ACB system

(Vector Labs). Non-radioactive in situ hybridizations were performed on 40!μm-thick

vibratome sections mounted on superfrost slides (Fisher) using reported methods(31).

Riboprobe for Pdgfrα was a gift of W.D. Richardson (University College, London);

riboprobe for Plp1 was a gift of J.D. Macklis, (Harvard University). For Golgi-Cox

impregnation stainings, adult brains (3-4 months old) from C57Bl/6 mice were processed

using the FD Rapid GolgiStain kit following manufacturer’s instructions (FD

NeuroTechnologies). 200 μm-thick coronal sections were prepared using a vibrating

microtome. Pyramidal neurons located in layer II/III and V-VI were randomly selected

and ImageJ 64 software was utilized to measure their position within the cortical wall

(distance between the pial surface and the center of the neuron soma) and the length of

their PMAS. For Black gold II stainings, 40!μm-thick vibratome sections were mounted

on superfrost slides (Fisher) and processed following manufacturer’s protocols (Histo-

Chem Inc.). Briefly, after rehydration, slides were incubated in 0.2% Black gold II in

0.9% saline for 12–15 min at 60°C. After washing in distilled water, slides were fixed in

sodium thiosulfate solution for 3 min at 60°C and then rinsed in distilled water,

dehydrated through graded alcohols (50%,70%,100%), cleared in xylene and mounted in

DPX (Sigma). For Gallyas stainings, adult macaque and human cortices were processed

as previously described(32). Briefly, 20 μm-thick cryostat sections (human tissue) or

40!μm-thick vibratome sections (macaque tissue) were washed in ddH20, passed through

a series of graded acetic acid steps, and then incubated in silver iodide solution (1% silver

nitrate) for 45' at room temperature. All tissue sections were imaged using a Nikon 90i

fluorescence microscope equipped with a Retiga Exi camera (Q-IMAGING) and

analyzed with Volocity image analysis software v6.0.1 (Improvision).

Human and macaque cortical tissue Specimens of adult human somatosensory cortex (medial postcentral gyrus) were

obtained from the collection of human brains of the Institute of Forensic Medicine at the

University of Turin, Italy. Specimens were fixed in 4% paraformaldehyde in phosphate

buffer (PB) 0.1 M, pH 7.4 for four hours at 4°C, and cryoprotected overnight in a 30%

sucrose solution before freezing, as previously described(33). 20 μm-thick sections were

cut on a cryostat. Specimens of adult Rhesus Macaque (Macaca mulatta) somatosensory

cerebral cortex (medial postcentral gyrus) were from brains described before. Briefly,

animals were deeply anaesthetized with ketamine (5–10 mg/kg, i.m.) and metomidine (30

μg/kg, i.m.) and perfused transcardially with isotonic saline followed by 4%

paraformaldehyde in 0.1 M PBS. Animal surgery, preoperative, and postoperative care

were performed according to Italian (DL.vo 116/92) and European (Directive 86-609 EU)

guidelines for experimentation on primates.

Cell quantification

For quantification of OPCs and OLs, anatomically matched sections within the

somatosensory cortex were processed to detect Pdgfrα and APC (n= 2 mice per marker;

4 sections, 6-8 hemispheres per brain). Boxes of 300 pixels in width and spanning the

thickness of the cortex were superimposed at matched locations on each section and

divided into ten equally-sized bins. Cells were manually counted in each bin, and bin-

distribution was defined as the percentage of cells in each bin relative to the total number

of cells.

Statistical analysis

All data are reported as the mean!±!s.e.m.. Normally distributed data were analyzed using

a Student’s t-test. For data that were not normally distributed a Mann–Whitney U-test

was used.

Gallyas Nissl

Gallyas Nissl

Mac

aque Hu

man

II/III

IV

V

VI

I

MBPwild type ( 1.5 year )

A

B

giulio srubek tomassyFig. S1

giulio srubek tomassy

(A) Histological staining of myelin by Gallyas on coronal sections of macaque and human sensory corticesshowing graded myelin distribution. (B) Immunohistochemistry for MBP on a coronal section of a 1.5 year old wild type mouse. Scale bars, 400 μm (A), 200 μm, (B).

giulio srubek tomassyFig. S1. Reduced levels of myelin in the upper layers are conserved in macaque and human neocortex and maintained in aged mice.

W= 500 m

D=60m

S1 Layer IV Layer VI

V1 Layer II/III

I

II/III

IV

V

VI

% m

yelin

cov

erag

e/to

tal a

xona

l len

gth

0

20

40

60

layer

VI

S1 lay

er IV

S1 lay

er II/I

II

V

1

***

***

A B

giulio srubek tomassyFig. S2

giulio srubek tomassyFig. S2. Different longitudinal coverage by myelin along axons of upper and deep layer pyramidal neurons in the adult mouse neocortex.

giulio srubek tomassy(A) Schematic of S1 dataset location in the mouse brain and representative magnifications of layer VI and IV. Also shown is a representativemagnification of layer II/III from the V1 dataset. (B) Percentage coverage (mean±s.e.m.) by myelin of axons in layers VI, IV (S1 dataset), and layerII/III (V1 dataset). Scale bars, 20 μm.

II/III

IV

I

giulio srubek tomassyFig. S3

giulio srubek tomassy Fig. S3. Relative distribution of pyramidal neurons reconstructed in layer II/III of V1 dataset.

giulio srubek tomassyRendering of reconstructed neurons and relative location on one representative image from the V1dataset. Scale bar, 50 μm.

Distance from pia ( m)1000800600400

*

***

giulio srubek tomassyFig. S4

giulio srubek tomassyFig. S4. Longitudinal profiles of myelination on pyramidal neurons of the deep cortical layers.

giulio srubek tomassyHigh-resolution renderings of myelin distribution along single axons of 12 layer V-VI pyramidal neurons traced and reconstructed in the S1 dataset. Myelin is rendered in white. Long unmyelinated tracts are indicated by asterisks. Scale bar, 50 μm.

1234 5

67

8

9

10

11

12

1 2

3 4

5 6

7 8

910

11 12

BA C

1

23

1

2

3

4

4

123

456

7

8

12

34

56

7

8

giulio srubek tomassyFig. S5

giulio srubek tomassyFig. S5. Distribution of synapses on representative neurons in layer II/III of the V1 dataset axons.

giulio srubek tomassyHigh resolution renderings of (A) an intermittent, (B) an unmyelinated and (C) a "long PMAS" neuron of the V1 dataset. Insets show all traced synapses.Red and green dots indicate the native position of each numbered synapse along the axons. Red, afferent synapses, green, efferent synapses. Scale bars, 50 μm (renderings), 0.5 μm (insets).

Table S1. Spatial coordinates of axon hillocks of each neuron reconstructed in layer

II/III of the V1 dataset. For best visualization of axons the zoom level should be set at 1.

# neuron x y z Intermittent 54304 59560 134 Intermittent 62340 56800 152 Intermittent 52640 43844 712 Intermittent 86748 56916 536 Intermittent 84240 48096 560 Intermittent 98596 53488 530 Intermittent 55804 51224 206 Intermittent 91692 62112 576

Unmyelinated 65968 44788 180 Intermittent 117312 75372 878 Intermittent 89212 75376 864 Long PMAS 87944 64516 822

Unmyelinated 98340 51060 892 Long PMAS 116304 60704 838 Intermittent 70680 66920 306 Intermittent 58824 66696 362 Intermittent 61444 48764 202 Intermittent 56776 64808 610 Intermittent 80624   74056   942  Intermittent 43976   68784   714  Intermittent 97388   66480   960  Long PMAS 67112   43244   406  

Table S2. Quantification of all traceable synapses along each axon labeled in the V1

dataset. For best visualization of synapses the zoom level should be set at -1.

# neuron (myelination

profile)

# afferent synapses

# efferent synapses

Intermittent 3 0 Intermittent 6 0 Intermittent 6 0 Intermittent 2 0 Intermittent 5 2 Intermittent 1 2 Intermittent 4 0 Intermittent 2 0

Unmyelinated 3 0 Intermittent 3 0 Intermittent 1 0 Long PMAS 4 1

Unmyelinated 9 3 Long PMAS 3 1 Intermittent 6 0 Intermittent 3 1 Intermittent 7 1 Intermittent 5 0 Intermittent 1 0 Intermittent 2 0 Intermittent 8 1 Long PMAS 6   0  

Movie Captions

Movie S1. Movie through the XYZ-axes of the V1 dataset showing reconstruction and

tracing of one intermittently myelinated neuron.

Movie S2. Movie of navigation along the Z-axis through 75 slices in layer VI of the S1

dataset.

Movie S3. Movie of navigation along the Z-axis through 75 slices in layer IV of the S1

dataset.

Additiional Data (separate files) Movies S1 to S3.

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1249766.Tomassy.SM.pdfSuppl1.pdf1249766fig. S1.pdf1249766fig. S2.pdf1249766fig. S3.pdf1249766fig.S4.pdf1249766fig.S5.pdfSuppl3.pdf