STEPS FOR SETTING UP COMPUTER SIMULATIONS USING "NEURON" ON CELLS RECONSTRUCTED USING "TREETRACE" MACROS FOR NIH-IMAGE




These are instructions for converting a neuron reconstructed from confocal microscope serial sections into a form which can be used for the simulations by the program NEURON, written by Mike Hines.

**TRACING/DIGITIZATION--one must first take the confocal photo slices of the neuron and trace them (digitize them) using NIH Image and the TreeTrace Macros (see cheat sheet or full documentation in the Treetrace manuals).

**TRANSFERRING NIH IMAGE MEASUREMENTS FROM THE MAC TO THE PC These instructions assume you have an IBM-PC Clone running NEURON 3.2.3. They also assume you have a copy of MegaEdit, or comparable text editing software, which will allow you to add line feeds to each Macintosh line terminated with a carriage return, and size the columns to provide a field-termination character (tab or space) between columns.

(1) When on the Macintosh, after you have completed the last trace, go to the file menu and select "export measurements." This will create a file with a .mea extension. Then transfer the .mea file to the IBM-PC clone (via floppy transfer, network transfer, etc.)

(2) Open the file using MegaEdit.

(3) Do a "Save As" from the file menu and store the file in the Windows folder you will be using for storage of your NEURON-compatible files. Exit MegaEdit.

**BASIC PROGRAM Once you have the data in PC-readable format, then you must make the preliminary template file for all of the computer runs.

(1) Make sure you have noted the diameter of the terminal axon section(s), and converted the pixel value into a micron value.

(2) Decide how many microns you want to extend the axons beyond the last digitized segment (for an "infinite" extension, 3l is usually sufficient). Calculate using the following equation, or use AXOCALC. 

				 3 x (Rm x a/2 x Ri)^1/2
"a" is the axon diameter, Rm is the specific membrane resistance (50,000), and Ri is the specific axoplasmic resistance (60). If you use AXOCALC to get a value for l, remember to convert from centimeters to microns. IMAG2HOC.exe (see steps below) requires the value to be in microns.

(3) After gaining this information from AXOCALC, activate IMAG2HOC.exe (you may exit any time from the application be typing ^c.

(4) The program will prompt you for certain values and parameters as follows:

INPUT FILE NAME, OUTPUT FILE NAME?
[type the name of the file from MegaEdit, followed by a comma, space, then the name of what the basic template file will be.

INPUT SESSION FILE NAME (OPTIONAL)?
[In the Interviews version of Neuron (currently 3.2.3 at the time of this writing), one has the option of appending a session file to the end of the .hoc file in order to change the appearance and placement of the windows. IMAG2HOC.exe will allow you an opportunity to append a session file to the .hoc file.]

Z-AXIS SPACING IN PIXELS?
A pixel value for the spacing between the slices of the confocal microscope stack.

PIXEL-SIZE IN MICRONS?
A micron value for pixel size.

NUMBER OF SEGMENTS?
This number reflects how many digitized sections are in the entire reconstructed neuron.

EXTEND NEURITES BY ADDING (um)?
If you wish to extend the neurites beyond the last digitized segment, type in a value (in microns) .

NEURITE EXTENSIONS TO BE DIVIDED INTO HOW MANY SEGMENTS (10)?
Input how many segments to divide the neurite extensions into.

ENTER TERMINAL SECTION-IDS OF 3 AXONS (IF <3, ENTER 0'S)?
IMAG2HOC.exe allows you to designate 3 axons. If there are less than 3 axons, you must type zeros for the other axons. For example, if the neuron has one axon, section 956, one would respond to this question by typing: 

		956,0,0
EXTEND AXONS BY ADDING (um)?
If you wish to extend the axons, input a value (in microns) here. 3l is usually sufficient for an infinite extension.

AXON EXTENSIONS TO BE DIVIDED INTO HOW MANY SEGMENTS (10)?
Input how many segments to divide the axon extensions into.

The program will now create the preliminary template file. This will include connecting all of the sections and creating the morphology of the cell, adding the xopen line that is necessary to load the graphical interface of NEURON 3.2.3, and adding a session file if you choose. Exit IMAG2HOC.exe.


**TO EDIT .tpl FILE AND INPUT DATA NEEDED FOR nrn
You now have an opportunity to add various comments to the .hoc file. For example, you might wish to enter comments about the type of cell that was reconstructed, basic info about the particular run you are going to do, etc. You can either open the file in MegaEdit, or type "EDIT [cellname].hoc" at the MS-DOS prompt.