MEASUREMENT CONDITIONS TO USE EGFP AS PROBE OF INTRACELLULAR PH USING FCS

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Although the indoleamine serotonin (5-hydroxytrypamine, 5-HT) has been shown to be involved in a multitude of proccesses in living organisms, the best known being it's role as a neurotransmitter, it is not the sole intrinsic indoleamine in the central or peripheral nervous system. For example, 5-methoxytryptamine (MT), tryptamine and n-acetyl-5-methoxytryptamine (melatonin) all are found in high concentrations in the nervous system and all are 3 photon accessible. We have begun to characterize the MPE fluorescence properties (excitation cross sections and photobleaching parameters) of various indoleamines including 5-hydrodroxytryptophan (5-HT's prescursor), 5-hydroxyindole acetic acid (the principal metabolite of 5-HT), 5-hydroxtryptophol (another metabolized form of 5-HT), in addition to the indoleamines mentioned above. All of these indoleamines are roughly equally fluorescent at equal concentrations and would be observable in vivo in the same wavelength range as 5-HT. Alth ough this makes it impossible to discriminate between 5-HT and it's various metabolites when imaging intrinsic tissue fluoresecence in the 340 to 380 nm range, it does greatly improve the chances of in vivo imaging of serontonergic neurons and we have recently successfully imaged the indoleamine autofluorescence in cell bodies freshly removed mouse pineal glands, mouse brain slices, mast cells and brain slices from an adult moth (Manduca sextus). The indoleamines listed above are all naturally occurring, non-aberrant compounds. Perhaps more importantly is the detection of deviant indoleamines created by abnormal oxidation chemistry of 5-HT and related compounds. Aberrant oxidation products of indoleamines have been implicated in the etiology of neurodegenerative diseases such as Alzheimer's disease and several psychotic diseases such as schizophrenia and major depression. In addition, oxidation products of 5-HT are formed after the administration of amphetamines and may be involved in the neurodegenerative effects of prolonged usage of such drugs. Many of these of the aberrant oxidation products of 5-HT appear to undergo rapid dimerization resulting in autofluorescent products with absorption bands in the 320 to 360 range and strong fluorescence in the 420 nm region and/or trimerization to form green fluorescent products that absorb in the 420 - 460 nm region. Both of these polymer forms are easily separable from the "norm al" indolamine fluorescence at 350 nm and allows for the measurement of the oxidative state of the indolamine pools within a cell using three photon excitation of 5-HT and other normal indoleamines with simultaneous two photon excitation the blue fluorescent dimers or green fluoresecent trimers formed by abnormal oxidation. This technique is currently being applied to in vivo studies of these important aberrant compounds.