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Sulzer lab downloads, tutorials, and supplemental material

1. Cell cultures

This booklet details our substantia nigra and VTA neuronal culture protocols used for neurodegeneration and neurotransmission studies.

This short set of instructions details our methods for chromaffin cell culture.

2. Tutorial on random walks and Schmitz/Sulzer modified random walks for simulations of electrochemical detection of neurotransmitter release

For an intro that explains random walks and how to design a random walk spreadsheet, read

Chapter 1 introduction to using spreadsheets to simulate random walks

To learn how to integrate Michealis-Menten kinetics for uptake transporters in a random walk (Schmitz/Sulzer model) read

Chapter 2.Schmitz/Sulzer random walk

To use the model, we have prepared the following instructions and Excel spreadsheets. First, download Chapter 3 and follow the advice.

Chapter 3. Using the spreadsheets

To download the Microsoft Excel spreadsheets designed to simulate amperometry or cyclic votammetry in the acute slice, download

xls. files (try these first; we find they often work best for PC downloads)

Random walk model for amperometry (.xls file)

Random walk model for cyclic voltammetry (.xls file)

(new as of January 2006) amperometry spread sheet with flux through a pore model (.xls file)

binhex files (works well for Macintosh)

Random walk model for amperometry (binhex)

Random walk model for cyclic voltammetry (binhex)

the binhex files require "unstuffing" to be used. The program "Expander" can be downloaded for free from http://www.aladdinsys.com/downloads/index.html

3. Information on neuromelanin isolation, identification, and breakdown

Details on EPR for neuromelanin detection

the following Quicktime videos by Kester Phillips show microglia phagocytosing and degrading neuromelanin in culture in timelapse video

Here is a differential interference contrast video of ventral midbrain / astrocyte / microglial coculture which shows a microglial filopodium grabbing a neuromelanin particle for phagocytosis. The frames advance by one image per two minutes.

Here is another DIC video in which a small microglial cell attaches to a neuromelanin particle, appears to activate nearby microglia, and then migrates with the particle towards a group of other microglial cells that proceed to destroy the particle. Frame rate as above.

A bright field video (.mov prepared in QuickTime) or here in mpeg (mp4) which shows the degradation, i.e., loss of pigment better than DIC optics, although without DIC's morphological detail.

4. Analysis of quantal release events in an Igor XOP
(written by Eugene Mosharov: e-mail Eugene)

Requires Igor Pro version 4.07 or later.

This analysis program can be used to detect and characterize amperometric spikes recorded from chromaffin cells, PC12 cells, neurons, etc. The following parameters can be calculated:

Peak parameters:
Amplitude (Imax, pA)
Duration (t1/2, ms)
Charge (Q, pC or # molecules)
Interspike Interval (ms)

Peak rising phase parameters:
Slope (pA/s)
Time to Peak (tP, ms)

Peak falling phase parameters:
(Can be fit by linear, exponential or double-exponential regressions)
Duration (ms)
Decay time constants (tau1 and tau2, ms)

Peak foot parameters:
Amplitude (Ifoot, pA)
Duration (tfoot, ms)
Charge (Qfoot, pC)

Click here to download the file in "ipf" (Igor Pro) format. The current version of the program is 8.20.

This is an ipf file (Igor Procedure File) for Igor Pro (has to be version 4.07 or later). To install the routine, unzip the archive and put the file Quanta_Analysis_ver.ipf into the Igor Pro/Igor Procedures folder. After starting Igor, you should see a bookmark "Prepare for quanta analysis" under the Macros menu. Clicking it creates windows and controls for the program. (As the program was written on a PC, the sizes of panels and windows may look odd on a Mac.) Many tune-ups in the program (on the topmost Menu panel) change how the data are analyzed and presented.

If you have questions about the program, or can suggest improvements, please e-mail Eugene Mosharov (em706@columbia.edu).

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