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      Label-free imaging of neurotransmitters in live brain tissue by multi-photon ultraviolet microscopy

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          Visualizing small biomolecules in living cells remains a difficult challenge. Neurotransmitters provide one of the most frustrating examples of this difficulty, as our understanding of signaling in the brain critically depends on our ability to follow the neurotransmitter traffic. Last two decades have seen considerable progress in probing some of the neurotransmitters, e.g. by using false neurotransmitter mimics, chemical labeling techniques, or direct fluorescence imaging. Direct imaging harnesses the weak UV fluorescence of monoamines, which are some of the most important neurotransmitters controlling mood, memory, appetite, and learning. Here we describe the progress in imaging of these molecules using the least toxic direct excitation route found so far, namely multi-photon (MP) imaging. MP imaging of serotonin, and more recently that of dopamine, has allowed researchers to determine the location of the vesicles, follow their intracellular dynamics, probe their content, and monitor their release. Recent developments have even allowed ratiometric quantitation of the vesicular content. This review shows that MP ultraviolet (MP-UV) microscopy is an effective but underutilized method for imaging monoamine neurotransmitters in neurones and brain tissue.

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          Most cited references 79

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          In vivo three-photon microscopy of subcortical structures within an intact mouse brain

          Two-photon fluorescence microscopy (2PM) 1 enables scientists in various fields including neuroscience 2,3 , embryology 4 , and oncology 5 to visualize in vivo and ex vivo tissue morphology and physiology at a cellular level deep within scattering tissue. However, tissue scattering limits the maximum imaging depth of 2PM within the mouse brain to the cortical layer, and imaging subcortical structures currently requires the removal of overlying brain tissue 3 or the insertion of optical probes 6,7 . Here we demonstrate non-invasive, high resolution, in vivo imaging of subcortical structures within an intact mouse brain using three-photon fluorescence microscopy (3PM) at a spectral excitation window of 1,700 nm. Vascular structures as well as red fluorescent protein (RFP)-labeled neurons within the mouse hippocampus are imaged. The combination of the long excitation wavelength and the higher order nonlinear excitation overcomes the limitations of 2PM, enabling biological investigations to take place at greater depth within tissue.
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            Release of secretory products during transient vesicle fusion.

            Patch-Camp experiments have shown that fusion of secretory granules with the plasma membrane does not always occur as an all-or-none event, but can develop slowly in a fluctuating manner or can be transient. These observations suggested that release could be detected during such incomplete fusion events. To test this hypothesis we have combined patch-clamp measurements of the activity of single exocytotic fusion pores in beige mouse mast cells with the electrochemical detection of serotonin released during the exocytotic events. We report here that on fusion pore opening there is a small release of serotonin which is directly proportional to the pore conductance. We also show that a significant release occurs during transient fusion events. These results demonstrate, to our knowledge for the first time, release of a neurotransmitter from a secretory vesicle that did not undergo complete fusion.
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              Real-time measurement of transmitter release from single synaptic vesicles.

              Neurotransmitter release is mediated by Ca2+ dependent exocytosis of synaptic vesicles. Neither the amount of transmitter released from individual synaptic vesicles nor the kinetics of this process have yet been directly determined. Using carbon fibres as electrochemical detectors, we have measured release of the neurotransmitter serotonin from cultured neurons of the leech. This technique allowed us to monitor transmitter discharge from single synaptic vesicles as spike-like oxidation currents at high time resolution, providing new insight into the mechanism of neuronal exocytosis. Two types of signals were characterized, corresponding to exocytosis of small clear and large dense core vesicles present in these cells. A small vesicle discharges about 4,700 transmitter molecules with a time constant in the region of 260 microseconds, whereas large vesicles release their content of approximately 80,000 molecules with a time constant of about 1.3 ms. Release from both vesicle types is initiated rapidly, with a rise time of less than 60 microseconds, suggesting an abrupt opening of a preassembled fusion pore.

                Author and article information

                Neuronal Signal
                Neuronal Signal
                Neuronal Signaling
                Portland Press Ltd.
                December 2018
                03 December 2018
                : 2
                : 4
                Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India
                Author notes
                Correspondence: Sudipta Maiti ( maiti@ 123456tifr.res.in )
                © 2018 The Author(s).

                This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY).

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                Pages: 14
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