Glutamate release

F. Frank LeFever flefever at ix.netcom.com
Wed Jan 22 23:47:21 EST 1997


In <5c4p3q$21e at mserv1.dl.ac.uk> "Michael Duggan"
<michael.duggan at camr.org.uk> writes: 
>
>Dear All,
>
>We want to measure glutamate release from cultured dorsal root 
>ganglion neurons. We have had some tries with radiolabelled D-Asp 
>and glutamine labelling of the cells, but the results are not as good 
>as we would like them to be (variability in loading, lack of calcium 
>dependence for release etc.).
>
>We would, therefore, like to try measuring the release of  endogenous 
>glutamate. The question is which is the best way to measure this? We 
>have tried using glutamate dehydrogenase and NADPH fluorescence, but 
>the signal was at the limit of detection. Obviously we could increase 
>the signal by increasing the number/density of the cells but we are 
>going blind doing the dissections already!
>
>There are methods in the literature using HPLC of derivatised 
>(o-phthalaldehyde, for example) samples but before we invest in
enormously
> expensive fluorescent detectors, can anybody answer the following 
>questions:
>
>1.    Are the hplc methods more sensitive than the enzymatic methods? 
>       If so by how much?
>
>2.    What are the best derivatisation reagents for this technique?
>
>3.    Are there any hplc methods which would allow sensitive detection
by 
>       absorbance rather than fluorescence (we have a UV detector 
>       already, so this would be a lot cheaper)?
>
>4.    Does anybody have any other good ideas about how best to do 
>       this?
>
>Thanks for any help you can give us
>
>Mike and John
>
>Michael J. Duggan,
>Centre for Applied Microbiology and Research,
>Porton Down,
>Salisbury,
>Wiltshire,
>SP4 0JG,
>UK
>
>michael.duggan at camr.org.uk
>
>Tel 01980 612733
>Fax 01981 612100


Research at Helen Hayes Hospital included demonstration that
hiuppocampal damage by seizures did not have to involve anoxia but
simply excitotoxic damage (presumably by glutamate).  Possibly they
measured glutamate, or in any case might have ideas for you.  Don't
know his e-mail (might be in Society for Neuroscience directory), but
you could try FAX:  Robert Sloviter, Ph.D.
                    USA (914) 947-3350.

                    Helen Hayes Hospital
                    West Haverstraw, NY 10993 USA

Frank LeFever
New York Neuropsychology Group



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