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The Cortical Box Method Manual

Junya Hirokawa

National Institute for Basic Biology

Division of Brain Biology

(Yamamori lab)

38 Myodaijicho

Okazaki, 444-8585

Japan

October 06, 2008

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Contents

1 Overview of Cortical Box Method......................................................................................................... 3

2 Download ............................................................................................................................................. 4

3 Requirements ........................................................................................................................................ 4

4 Installation ............................................................................................................................................ 5

5 Disclaimer............................................................................................................................................. 5

6 Manual for Cortical Box Method........................................................................................................... 6

6.1 Flow chart for analysis by Cortical Box Method ............................................................................. 6

6.2 Preparation of serial coronal sections.............................................................................................. 7

6.3 Preprocessing ................................................................................................................................. 8

6.4 Standardization of cortical sections by CxStand.............................................................................. 9

6.5 Construction of 3D cortical box and 2D layer maps ...................................................................... 14

Appendix ............................................................................................................................................... 15

References ............................................................................................................................................. 17

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1 Overview of Cortical Box Method

To comparatively investigate many brain sections used for different stainings (e.g., Nissl staining, c-Fos

immunohistochemistry, in situ hybridization, neuronal tracer dye) and/or those obtained from different

animals, we need to transform data into a standard format for integration. The Cortical box method is a

way to standardize the serial coronal sections of the rat cortex. It allows us to transform the image of a

cortical section into a standard stripe and assemble those stripes into a 3-D box.

There are four main features of this method. (1) One can grasp the spatial distribution pattern of the

whole data at a glance. This is important to understand a lot of samples comparatively. (2) The method

sets the cortical layer dimension in one axis. It allows us to analyze the layer specific patterns that are

important in understanding the cortical structure. (3) In addition to qualitative analysis, standardized 3-D

matrix is useful for quantitative and statistical analysis. (4) The algorism for standardization is simple

and straightforward, although the process requires some manual processing. Many differently processed

data can be potentially integrated by our standardization, which could be useful to correlate cortical

functions with defined structures. Thus, we welcome other researchers to use this method.

Repercussions using this method can be seen in the Ref.1 for functional mapping using c-Fos

immunohistochemistry and Ref.2 for gene expression pattern analysis using in situ hybridization.

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2 Download

Programs and example files can be downloaded from our web site (http://www.nibb.ac.jp/cortex/).

CxStand ver1.0 (for standardization of rat cortex, described in chapter 6.4)

Sample files (17 JPEG images, ISH for RORb gene described in Ref. 2)

These sample files were uploaded so that users can test our standardization program. Unzip the

downloaded file. It contains two folders named "ORI" and "SKL", which are explained in the chapter 6.4

in this manual. File name "01.jpg" corresponds most rostral cortical section (-2.1 mm from bregma) and

17.jpg corresponds to most caudal cortical section (-6.3 mm from bregma).

3 Requirements

Cortical Box Method software is written in Labview and requires Labview version 7.0 (or later) and

Vision development version 7.0 (or later). Labview and Vision are products of National Instruments

(http://www.ni.com/ ). I confirmed (*) the Cortical box software works in the latest versions of

Labview (ver.8.6) and Vision depelopement (ver. 8.6), which can be downloaded without charge in the

National Instruments web site (http://www.ni.com/) and can be used as evaluation softwares for 30 days.

CxStand has only been tested on PCs running the Windows family of operating systems.

*A VI called "image to image" does not exist in Labview 8.6, please replace it into "image to image 2".

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4 Installation

Unzip the downloaded files in any location of the hard disk. This software does not modify registry.

Please show extensions of the file names (e.g., 01.jpg), if they are hidden.

5 Disclaimer

This code is copyright © by the original author (JH), 2008.

None of the authors nor their institutes assume any liabilities for this code. Use at your own risk. We have

done our best to ensure that this code is correct, but do not make any guarantees. This program is

distributed in the hope that it will be useful, but without any warranty; without even the implied warranty

of merchantability or fitness for a particular purpose.

Use of this code should be acknowledged in any paper that uses data analyzed with it.

Acknowledgement should be in the methods section citing Ref. 1 and/or Ref. 2. This code may be

distributed freely. Modifications of the original source code is allowed without permission but please

inform me (JH) if the modifications are useful.

Send bug reports, questions, and comments to the author (JH, cortex@nibb.ac.jp). I will do my best to

fix bugs, answer questions, incorporate new features, etc, if I have time.

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6 Manual for Cortical Box Method

6.1 Flow chart for analysis by Cortical Box Method

Taking pictures of serial sections

Preparation of serial coronal sections

Preprocessing (density map for single cell resolution analysis)

Standardization of cortical sections by CxStand

Construction of 3D cortical box and 2D layer maps

Color maps, statistical analysis, etc…

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6.2 Preparation of serial coronal sections

6.2.1 Tissue preparation

We use male Sprague-Dawley rats (~400 g). Special care was taken to maintain the orientation of sections.

We use a silicon template to cut vertically the brain into half (anterior and posterior parts). Coronal

sections from both hemispheres were cut at 40 m thickness using a freezing microtome. Every seventh

section was preserved. These sections cover the posterior part of one rat brain hemisphere with an

approximately 280 m interval (-2.1 to -6.3 mm from Bregma). Every sections used in this analysis

should not contain any artifacts such as tearings, bubbles and derrises in the target cortical part.

Therefore, staining and inclusion should be very carefully conducted.

6.2.2 Image acquisition.

For Nissl staining and ISH staining, digital images (1360 x 1024 pixels) were captured using the 1.25 x

objective in the gray scale with 8 bits (10.3 m/pixel). Smaller and larger sizes than 1360 x 1024 pixels

should work but the parameters such as "dividing bins" should be reconsidered.

For the analysis that needs single cell resolution such as c-Fos immunohistochemistry, we take higher

resolution images using the 4 x objective and reduce their pixel sizes by following preprocessing.

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6.3 Preprocessing

For Nissl staining and some of the ISH stainings, the sections have high signal to noise ratio and the

overall patterns can be captured even in lower resolution. In these cases, this preprocessing is not

necessary. For cortical box analysis that needs higher resolution (e.g., c-Fos immunohistochemistry,

staining for tracer dyes), the detection of each signal at cellular resolution is necessary. For this purpose,

higher resolution images of each parts of cortex were captured and reconstructed into a single image.

Then, the image was processed to be a "density map" that is no longer high resolution. Details are

described in Ref. 1.

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6.4 Standardization of cortical sections by CxStand

6.4.1 Standardization of cortex

CxStand ver.1.0

The goal of the image processing by CxStand software is to transform a part of cortex into a standard

rectangular form (Standardized cortical section). The "Batch_CxStand" software repeats this process to a

set of serial sections to create a Cortical Box.

To use CxStand software, you need to create two folders named ORI and SKL in the same directory.

You need to prepare files in those folders as explained below. Here, I explain how to prepare those files

by taking an image of a serial section stained by RORbeta ISH as an example. A set of images used here

can be downloaded from the link of example files above.

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6.4.2 Preparation of ORI files

An ORI file is an image of which cortical part is extracted and transformed into a standardized rectangular by the CxStand software.

The program only works when a hemisphere is right-sided (please flip horizontal, if not) and its

position should be approximately centered and the angle approximately upright (it needs not to be

exactly positioned).

The original image has shadowing effect by light source. To eliminate such effect, the background image is captured on the same condition.

The background image is subtracted from the corresponding ORI file. No further processing should be performed on this image. The resulting image is saved in the folder named "ORI". Fos c-Fos immunohistochemistry, "c-Fos density map" was used here instead.

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6.4.3 Preparation of SKL files A SKL file is an image that defines a part of cortex in ORI image to be processed. For CxStand software Ver.1.0, users have to define it manually.

To create a SKL file, the ORI file is used. I usually do this manual step using Adobe Photoshop. To better visualize the section structures, change the contrast and/or make a pseudocolor image of the ORI image. Be careful not to over-write the original ORI file at this step.

By using polygon selection tool, trace the part of cortex (The detail is explained in Appendix). Then, delete outside of the selected region.

Finally, fill inside of the selected region black and save this image in the directory of "SKL" with the name of the same serial number of the corresponding ORI file. The pixel size of the image must be the same as that of the corresponding ORI file (1360 x 1024 pixels in the RORb example). This process can be semi-automated using action script and batch processing function of Photoshop.

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6.4.4 Running CxStand

Open "Batch_CxStand.vi" in the "CorticalBoxSoftwares" directory.

Then, Run the program by pressing the arrowhead icon.

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Choose "ORI" folder by pressing "select button"

The program automatically processes the data and stop in the end. Processing time depends of machine

but it will end within a few minutes. You can also monitor the status in the "CorticalBox" window (red).

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The results of processing will be seen in same folder that has "ORI" and "SKL" folders.

"SECT" is a folder that has information of transformation rate of each image as text files.

"STAN" is a folder that has the standardized cortical sections of ORI images as JPEG files.

You can handle the above "STAN" folders as an assumed cortical box. Just aligning those files will be a

"flattened cortical box". Thresholding and pesudo-coloring them will also be helpful to grasp the whole

pattern.

6.5 Construction of 3D cortical box and 2D layer maps

To create 2D layer map, the spaces between them were linearly interpolated to reconstruct 3D

organization. To generate the standardized map of a particular layer (1000 x 340 pixels) (width and

Bregma distances, respectively), the specific layer fraction (10-30 % for layer 2/3; 30-50% for layer 4;

50-75 % for layer 5; 75-100 % for layer 6) was extracted from the standardized cortical box and

compressed into a two-dimensional map by averaging. Post hoc smoothing (spatial averaging) was

achieved using a moving window operator (41 x 41 pixels).

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Appendix

Selection of a part of cortex Here, I explain how to select a part of cortex manually for Cortical box method (to make SKL files) in detail. It consists of selections of four borders; outer contour (OC, pial surface), inner contour (IC, border between white matter and gray matter), mediodorsal end (MD, corpus callosum) and lateroventral end (LV, rhinal sulcus).

Definition of Outer Contour (OC) The pial surface is traced ignoring and compensating small tearing or cracks as shown in right figure. This process could be automated but the line should be smooth.

Definition of Inner Contour (IC) The difficulty to select IC border depends on the kinds of staining. Especially when there are no signal in layer 6 (e.g., RORb for ISH), it is hard to delineate the border automatically (right figure). But it is possible for human to draw a border by compensating around information.

Definition of Latero-Ventral border (LV) I set LV border so that the border line is perpendicular to layers and it also needs to be set on the center of the rhinal sulcus.

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Definition of Medio-Dorsal end (MD) MD border line is set so that it is perpendicular to layers and it also need to be set on the apex of the corpus callosum shown by arrows in the following figures. I show three typical cases of definition of MD border since the shape of corpus callosum is slightly different at the positions from Bregma. The numbers on the left figure indicate the distances (mm) from bregma.

Target region of Cortical Box method The way we select MD and LV borders sets limit to the range of cortex that can be used for this method.

Since LV border relies on the rhinal sulcus, it is difficult to define LV border in the rostral part of the

cortex, where the rhinal sulcus disappears (around 0 mm from Bregma). In addition to this limitation,

the selection of MD border limits caudal end. In the caudal cortical region (after approximately -8 mm

from Bregma), the white matter disappears depending on the angles of sections.

-5.1

-6.3

-2.1

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References 1. Hirokawa, J., Bosch, M., Sakata, S., Sakurai, Y. & Yamamori, T. Functional role of the secondary

visual cortex in multisensory facilitation in rats. Neuroscience. 153, 1402-1417 (2008) 2. Hirokawa, J., Watakabe, A. & Ohsawa, S. and Yamamori T. Analysis of Area-Specific Expression

Patterns of RORbeta, ER81 and Nurr1 mRNAs in Rat Neocortex by Double In Situ Hybridization and Cortical Box Method. PLoS. ONE. in press