Label-Free Dopamine Imaging in Live Rat Brain Slices

Abstract

Dopaminergic neurotransmission has been investigated extensively, yet direct optical probing of dopamine has not been possible in live cells. Here we image intracellular dopamine with sub-micrometer three-dimensional resolution by harnessing its intrinsic mid-ultraviolet (UV) autofluorescence. Two-photon excitation with visible light (540 nm) in conjunction with a non-epifluorescent detection scheme is used to circumvent the UV toxicity and the UV transmission problems. The method is established by imaging dopamine in a dopaminergic cell line and in control cells (glia), and is validated by mass spectrometry. We further show that individual dopamine vesicles/vesicular clusters can be imaged in cultured rat brain slices, thereby providing a direct visualization of the intracellular events preceding dopamine release induced by depolarization or amphetamine exposure. Our technique opens up a previously inaccessible mid-ultraviolet spectral regime (excitation ~270 nm, emission < 320 nm) for label-free imaging of native molecules in live tissue.

Effect of amyloids on the vesicular machinery: implications for somatic neurotransmission

“…The majority of the vesicles, however, contain serotonin and are also stained by the FM dye, and probably belong to the recycling pool, at least at the relatively long time scale of somatic exocytosis. Though less evidence is available for somatic DA vesicles, the overall organization and the kinetics of exocytosis appear to be similar to those observed for the somatic serotonergic machinery [58]. While the effect of amyloids on the relative size of these somatic vesicular pools is not known, such experiments are, in principle, doable with the technology we have developed.…”

“…Somatic release has been observed in multiple systems, including serotonergic neurons such as the Retzius neurons of leech [45,51,52], mesencephalic trigeminal nucleus of rat [53], primary cultures of raphe neurons and raphe slices of rats [49,50,54], and serotonergic neurons obtained from differentiated human embryonic stem cells [55]. It has also been observed in DA neurons of rats and mice [56][57][58]. While the precise role of somatic exocytosis is unclear, it is hypothesized to mediate responses which persist for long durations, for example, mood changes [46].…”

“…Since both serotonin and dopamine fluorescence can be imaged directly using multiphoton excitation, it is possible to measure both the content and the population of monoamine-containing somatic vesicles in a given cell [49,50,54,58]. This presents a unique opportunity compared to studying synaptic exocytosis, as vesicles at a typical synapse cannot be visualized in live cells.…”

“…It is also possible to visualize the Ab 40 oligomers eventually entering the cells (figure 2b). Such imaging can, in principle, be combined with direct multi-photon imaging of serotonin [117] or dopamine [58]. Thus, some of the experimental limitations of the synaptic experiments can be overcome if a similar set of experiments are attempted in a cell carrying out somatic exocytosis.…”

Deep‐Ultraviolet Biomolecular Imaging and Analysis

“…They detected leukocytes with two‐photon excitation tryptophan autofluorescence of endogenous proteins. Maiti and co‐workers measured the dynamics of neural transmitters, such as dopamine and serotonin, in brain slices and cultured neural cells by autofluorescence at two‐photon excitations of DUV absorption bands.…”