I would like to check the functionality of neurons other than electro physiology and HPLC. Can anyone please help me? Thanks in advance.
Fast-scan cyclic voltammetry has been used in slice preparations to measure dopamine released by neurons. It will give you sub-second temporal resolution but it will be a relative value (i.e. you will measure a baseline current and then measure your differences from that) as opposed to an absolute concentration. You can calibrate your electrodes with known concentrations of dopamine to get a ballpark idea of how your current corresponds to dopamine concentration.
http://www.sciencedirect.com/science/article/pii/S0014299912003809
http://pubs.acs.org/doi/abs/10.1021/ac5008927
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3686531/
Voltammetry is suitable to measure dopamine. Maybe GABA is also measureable using biosensors.
Article Electrochemical responses of carbon fiber microelectrodes to...
If you have a relationship with the organic chemistry dep. you can try detecting it with NMR in culture media. Relatively fast and high-througput. A quick PubMed search should give you an idea of their MR spectra.
That being said, I think microdialysis with HPLC is probably going to give you better spacial/temporal resolution, specificity, and quantification than other methods.
Fast-scan cyclic voltammetry has been used in slice preparations to measure dopamine released by neurons. It will give you sub-second temporal resolution but it will be a relative value (i.e. you will measure a baseline current and then measure your differences from that) as opposed to an absolute concentration. You can calibrate your electrodes with known concentrations of dopamine to get a ballpark idea of how your current corresponds to dopamine concentration.
http://www.sciencedirect.com/science/article/pii/S0014299912003809
http://pubs.acs.org/doi/abs/10.1021/ac5008927
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3686531/
I'd like to inject a word of caution about using voltammetry, rather than HPLC, to detect dopamine in culture medium. As John mentioned, you won't directly measure dopamine concentration using this method. The electrodes must be postcalibrated, but electrode sensitivity generally declines over time - your postcalibration data will only give an estimate of dopamine concentration. The lab in which I learned voltammetry also used microdialysis and HPLC, for this exact reason. The big advantage of FSCV is its high temporal resolution, rather than its absolute accuracy. HPLC is the gold standard for a reason.
Alternative methods are Capillar Zone Electrophoresis couple to laser induced fluorescence and mass spectrometric assay for the simultaneous measurement of neurotransmitters. You can use a micro dialysis probe and collect small dialysates from your culture.
High temporal resolution monitoring of neurotransmitters, dopamine (DA), noradrenaline (NA), gamma-aminobutyric acid (GABA), glutamate (Glu), l-aspartate (L-Asp), can be achieved using microdialysis and capillary electrophoresis with laser-induced fluorescence detection (CE-LIFD) . The sampling time is limited by the minimal volume required for the analysis by the automated CE system used: neurotransmitters could be determined in 667 nl dialysates (940 nl after derivatization), i.e. in samples collected every 20 s with a flow rate of 2 microl/min. The detection limits at the dialysis probe were 3 x 10(-9), 1 x 10(-9), 1.9 x 10(-8), 4.2 x 10(-7), 2.1 x 10(-7) mol/l for DA, NA, GABA, Glu and L-Asp, respectively. The protocol was validated using in vitro/in vivo tests and the performances--repeatability, linearity, characteristics of the probes--were determined. (SeeJ Neurosci Methods. 2004 Dec 30;140(1-2):29-38)
Mass spectometry can be coupled to High-performance liquid chromatography/tandem (see Journal of Neuroscience Methods Volume 138, Issues 1–2, 30 September 2004, Pages 123–132)
Best
thanks for the precious replies.... bt i dnt have istruments for patch clamp assay. could any one suggest calorimetric using dyes or fluorescent compounds...
anyhow thanks for helping me
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