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neuroimaging - itracellular calcium measurement in neurosynaptosomes (Apr/19/2009 )

Dear all,

I have few problems regarding measurement of calcium influx in neurosynaptosomes. I am making the preperation by the method desribed in book"neurotransmitter methods", which involves a two stage filteration of brain homogenate , firstly with 100micron filter and again with 8 micron filter, the resulting suspension is belived to be containing neurosynaptosomes. Next the preperation is loaded with Fura2 AM and the resulting fluroscence measured.I need to know whether there is any method to check the viability of the neurosynaptosomes preperation as I`m not getting the desired activity. Is there any other method to prepare neurosynaptosomes.

It would be very kind to anyone of u who can help me in this regard.

Thank you

-vikasmishra01-

i haven't worked with synaptosomes in 25 years and can't locate the procedure that i used but i remember that it involved homogenization with loose pestle and differential centrifugation. the procedure is either written or referred in our publication:

Malik, M.N., M.D. Fenko and H.M. Wisniewski, Purification and Partial Characterization of Two Forms of Ca2+-Activated Neutral Protease From Calf Brain Synaptosomes and Spinal Cord, Neurochem. Res. 1984; 9: 233-240.

i also remember that synaptosomes are not stable and break down rapidly and are not storable.

-mdfenko-

Google did a great job for this one...

http://www.abcam.com/index.html?pageconfig...e&rid=10731

http://www.biochemj.org/bj/176/0365/1760365.pdf (from 1978! ... yields metabolically active synaptosomes, or so it claims)

http://www3.interscience.wiley.com/journal...=1&SRETRY=0 (also from 1984)

...there's many more out there.

-Carlton

-Carlton H-

I`m very thankful to the members MDFENKO and Carlton H who had provided such extensive literature on the preparation of synaptosomes, but I was looking for the preparation of neurosynaptosomes or also known as synaptoneurosomes. There is a difference between both the preparation synaptosomes contain predominantly persynaptic structures whereas synaptoneurosomes preparation is enriched with postsynaptic structures. And since the receptor whose activity I`m looking for is located on postsynaptic part the preparation of synaptoneurosomes will be much appropriate to work on.

I again thank all of U for your generous efforts.

-vikasmishra01-

Well, then... Allow me to give you a few more leads:

http://www.sciencedirect.com/science?_ob=A...076ba78064f87ca

Also, if your library has a copy of the book "Neurotransmitter Methods" you should be able to find a protocol in there.

Good luck!
-Carlton

-Carlton H-