Selective detection of neurotransmitters by fluorescence and chemiluminescence imaging

Abstract

In recent years, luminescence imaging has been widely employed in neurochemical analysis. It has a number of advantages for the study of neuronal and other biological cells: (1) a particular molecular species or cellular constituent can be selectively visualized in the presence of a large excess of other species in a heterogeneous environment; (2) low concentration detection limits can be achieved because of the inherent sensitivity associated with fluorescence and chemiluminescence; (3) low excitation intensities can be used so that long-term observation can be realized while the viability of the specimen is preserved; and (4) excellent spatial resolution can be obtained with the light microscope so subcellular compartments can be identified. With good sensitivity, temporal and spatial resolution, the flux of ions and molecules and the distribution and dynamics of intracellular species can be measured in real time with specific luminescence probes, substrates, or with native fluorescence. A noninvasive detection scheme based on glutamate dehydrogenase (GDH) enzymatic assay combined with microscopy was developed to measure the glutamate release in cultured cells from the central nervous system (CNS). The enzyme reaction is very specific and sensitive. The detection limit with CCD imaging is down to µM levels of glutamate with reasonable response time. We also found that chemiluminescence associated with the ATP-dependent reaction between luciferase and luciferin can be used to image ATP at levels down to 10 nM in the millisecond time scale. Similar imaging experiments should be feasible in a broad spectrum of biological systems.

Monitoring real-time release of ATP from the molluscan central nervous system

“…It is released from neurons simultaneously with other neurotransmitters and acts as both a fast transmitter and a neuromodulator (Brown and Dale, 2002;Jo and Role, 2002;Newman, 2003;Santos et al, 1999Santos et al, , 2003Von Kugelgen et al, 1994;Wang and Yeung, 2001). In this work, we applied our ATP imaging scheme, which has to date only been employed with cultured astrocytes and endothelial cells, to the detection of ATP release in the ganglia of the freshwater snail Lymnaea stagnalis.…”

Autofluorescence Spectrofluorometry of central nervous system (CNS) neuromediators

“…The main absorption and emission properties of 5-HT in an aqueous solution involve the UV, near-UV spectral region [16]. Excitation and emission properties of 5-HT at longer wavelengths have been already reported that, despite their weak signal, were considered for an application of near-UV-visible fluorescence microscopy to the in situ imaging study of the uptake and depletion of neurotransmitters in astrocytes [17][18][19][20]. The application of multiphoton excitation showed the appearance of a broad emission band centered at 505 nm, as a result of indoleamine photoionization [21].…”

Dual‐enzyme, co‐immobilized capillary microreactor combined with substrate recycling for high‐sensitive glutamate determination based on CE

“…Finally, this method was successfully applied to determine the glutamate content in rat plasma and serum samples. Since glutamate possesses few functional groups that can be exploited using conventional means of detection, enzymatic assay is an alternative approach to chemical derivatization procedures and produces a detectable analyte upon conversion of the substrate [3][4][5]. Typically, free enzymes are not stable for long time and their catalytic activities gradually decrease because of unfolding of its native structure caused by effects of heat and pH.…”