Multiphoton Autofluorescence Imaging of Serotonin

Herein, the autofluorescent imaging of serotonin in a rat brain slice is proposed to be evaluated with multiphoton excitation microscopy (MPM). If successful, this method could be used in conjunction with fluorescence correlation spectroscopy (FCS) to quantitatively and qualitatively assess real-time neurotransmitter dynamics in situ and in vivo.

Chemical neurotransmission relies on the signaled release of specific molecules from synaptic vesicles, which diffuse through the synaptic space and trigger a variety of post-synaptic responses. Hard data about neurotransmitter release, diffusion and re-uptake is essential for a better understanding of the brain, including such areas as behavior and neurodegenerative disease, yet this information has proven difficult to obtain. Many strategies have been employed to detect and quantify neurotransmitters and their dynamics in living tissue. Electrochemical methods are sometimes used, alone and in combination with microdialysis, HPLC or capillary electrophoresis. For instance, cyclic voltammetry at carbon fiber electrodes is used to sensitively detect real-time neurotransmitter release and provide concentration and kinetic data. However this method lacks spatial resolution; a carbon fiber tip is 7 microns whereas a typical neuronal synapse is much smaller, around 100 nm. lmmunohistochemistry has also been used to characterize neuron morphology and localization, but this data is not quantitative, and live tissue imaging is not possible. Confocal microscopy can be used to achieve the spatial resolution necessary to monitor neurotransmission events in the area of a single synapse. If time sensitive total-molecule concentrations and kinetic rates could be extrapolated with FCS, much useful information could be gained.

To use fluorescence microscopy, neurotransmitters are usually attached to fluorescent molecules capable of emitting photons upon excitation. This is often done, although the labeling procedures and/or necessary genetic manipulations can be difficult or time-consuming. In addition it is uncertain whether the addition of bulky fluorophores will significantly alter neurotransmitter base levels or dynamics. To avoid these problems, the natural fluorescent properties of certain molecules (endogenous fluorophores) can be exploited. Most biological studies with autofluorescence microscopy have focused on NADH (emission between 400-500 nm), which provides useful information about metabolism, cancer and other areas. In the UV region, tryptophan and its derivatives are a major source of autofluorescence, allowing for noninvasive visualization of skin, and muscle tissues, and blood vessel components.

One such tryptophan derivative is serotonin, a monoamine neurotransmitter that is active in many areas of the mammalian brain. Serotonin plays a role in many neurophysiological functions including the regulation of mood, anxiety, and appetite. It is thought to have a major impact on depression, and Alzheimer’s disease and aging, among other processes. Like its tryptophan precursor, serotonin exhibits one-photon absorption below 300 nm and emits around 340 nm. These wavelengths are in the ultraviolet spectrum making single photon excitation impractical in live tissue.'UV excitation cannot penetrate into tissue, instead causing damage and preventing reliable measurements. However, many important important neurotransmitters (including serotonin) exhibit autofluorescent behavior at UV wavelengths, providing an excellent means for their study if UV damage could be avoided. Multiphoton excitation of serotonin with light from lower frequency lasers has been shown to alleviate this problem, allowing for spatially resolved, 3D localization and visualization of fluorescing species in Iiving tissue. At these higher wavelengths, tissues exhibit far less scattering, providing better resolution at greater depths than is possible with single-photon excitation. This technique has allowed label-free imaging of serotonin in living neural and mast cell cultures, providing insights on localization, distribution and exocytosis.