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Label-free Ratiometric Imaging of Serotonin in Live Cells Anand Kant Das, Barun Kumar Maity, Dayana Surendran, Umakanta Tripathy, and Sudipta Maiti ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00132 • Publication Date (Web): 10 Aug 2017 Downloaded from http://pubs.acs.org on August 11, 2017
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ACS Chemical Neuroscience
Label-free Ratiometric Imaging of Serotonin in Live Cells
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$
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Bhabha Road, Colaba, Mumbai, 400005, India.
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†
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Dhanbad, 826004, Jharkhand, India.
Anand Kant Das$#§, Barun Kumar Maity$§, Dayana Surendran$, Umakanta Tripathy†,* and Sudipta Maiti$* Department of Chemical Sciences, Tata Institute of Fundamental Research (TIFR), Homi
Department of Applied Physics, Indian Institute of Technology (Indian School of Mines),
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ABSTRACT
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Ratiometric imaging can quantitatively measure changes in cellular analyte concentrations
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using specially designed fluorescent labels. We describe a label-free ratiometric imaging
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technique for direct detection of changes in intravesicular serotonin concentration in live
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cells. At higher concentrations, serotonin forms transient oligomers whose ultraviolet
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emission is shifted to longer wavelengths. We access the ultraviolet/blue emission using
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relatively benign three-photon excitation and split it into two imaging channels, whose ratio
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reports the concentration. The technique is sensitive at a physiologically relevant
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concentration range (10 to 150 mM of serotonin). As a proof of principle, we measure the
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increase of intravesicular serotonin concentration with the addition of external serotonin. In
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general, since emission spectra of molecules are often sensitive to concentration, our method
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may be applicable to other natively fluorescent intracellular molecules which are present at
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high concentrations.
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KEYWORDS: Multi-photon microscopy, neurotransmitter imaging, serotonergic neurons,
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quantitative imaging
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INTRODUCTION
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Monoamine neurotransmitters in the brain, such as the indoleamine serotonin (5-HT), are
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involved in the regulation of several processes, including mood, emotion, reward, anger,
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aggression, appetite, sleep and memory.1-3 Concentration of serotonin in different brain
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regions is affected by drugs of abuse, alcohol, stress, depression and several neurological and
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psychiatric conditions.4-7 Alteration of vesicular serotonin content is a marker for cellular
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differentiation8 and may also be related to neurodegenerative disorders.4 Hence, it is
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imperative to develop techniques which can accurately measure serotonin concentration
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changes. However, no direct imaging technique exists for accurately quantifying changes in
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intravesicular serotonin concentrations inside living cells.
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It is difficult to quantitatively determine analyte concentrations (or changes thereof)
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with conventional fluorescence reporters, because the amplitude of emitted signal is
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influenced by a variety of factors such as uneven loading of dye, intracellular
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compartmentalization of probe, effect of environment on the quantum efficiency, sample
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geometry etc.9 Vesicular size variations introduce another systematic error which makes
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intensity based imaging unsuitable as a measure of intravesicular concentration. Intensity
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based estimations show large apparent variations of concentrations between individual
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vesicles, simply because the larger vesicles are able to fill the probe volume, while smaller
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ones cannot (though they may contain the same concentration)10. However, if the shape of the
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fluorescence spectrum changes as a function of analyte concentration, then it should in
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principle be possible to carry out quantitative ratiometric imaging. Ratiometric imaging
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techniques have the advantage that the observed analyte-induced changes in the spectra
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provide an internal concentration reference. A wide variety of ratiometric sensors have been
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developed for imaging and sensing of various cations, anions and biomolecules and for
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various biomedical applications.11 If the analyte itself is fluorescent, then it may be possible
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to perform label-free ratiometry. Unfortunately, most intracellular fluorescent species are
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only excitable in the ultraviolet. We have earlier circumvented this problem and imaged
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intracellular neurotransmitters, such as dopamine and serotonin, by exploiting the principles
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of multi-photon microscopy (MPM).12,
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maps of serotonergic cells in rat brain slices,14 and allowed us to measure sequestration,10
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somatic release,15 and the dynamics of somatic exocytosis in serotonergic cells.16
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Interestingly, our earlier observation showed that as the serotonin concentration increases
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from sub mM to 100’s of mM, the emission spectrum exhibits a long wavelength tail which
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This technique allowed us to generate regional
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extends to the visible region.17 This is due to the formation of non-covalent oligomers which
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monomerize on dilution. The concentration changes result in an iso-emissive point in the
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normalized emission spectrum at 376 nm17 (see inset of Figure 2). Taking a cue from this,
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here we develop a ratiometric technique which can directly estimate intravesicular serotonin
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concentration changes in neuronal cells in culture.
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RESULTS AND DISCUSSION
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We first generate a calibration curve by recording signals from a series of
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concentrations of freshly prepared aqueous serotonin solutions (from 10 mM to 800 mM).
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The ratio of the long-wavelength (T) signal (> 360 nm) to the short wavelength (R) signal (
360 nm), while the other goes in the reflection (R) direction (UV signal < 360 nm).These signals are passed through homemade 1 cm thick CuSO4 solution filters to block the excitation light, and are detected by two analog photomultiplier tubes (PMTs, Model: P30A-01, Electron Tubes Limited, UK) after. We constructed an external non-descanned detection unit which houses the dichroic and these two PMT’s in an orthogonal geometry in a light-tight brass assembly (Figure 1). The ratio of the intensity of the two channels (reflection (R), channel A and transmission (T), channel B) can be measured, and this provides a direct estimate of the concentrations. We test this with an aqueous solution of serotonin (800 mM). The cubic excitation intensity dependence of the signal obtained shows that it is a result of 5 ACS Paragon Plus Environment
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three photon excitation (see supporting information, Figure S1). The experiment is performed at different concentrations of freshly prepared serotonin solutions in water and with RN46A cells under same imaging conditions. Finally, the histogram of the (T/R) ratios of the average signal intensity in the transmission to reflection channel is plotted. All imaging is performed at room temperature (~ 25ºC).
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Cell culture: Low passage cells of RN46A (rat serotonergic neuron-derived cell line) is
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cultured in DMEM-F 12 (1:1) (Gibco, USA) media supplemented with 5% FBS, 50 units/ml
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Penicillin and 50 µg/ml Streptomycin (Gibco, USA) at 39˚C (for differentiation) under
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humidified air containing 5% CO2 in T-25 canted neck flasks (Falcon, USA). Media is
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changed every 48 hours. For ratiometric studies, the cells are passaged on Poly-L-lysine
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(Sigma, USA, 0.1 mg/ml) coated homemade cover slip bottomed petridishes for 1 day.
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Imaging is performed in Thomson’s buffer (TB) (146 mM NaCl, 5.4 mM KCl, 1.8 mM
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CaCl2, 0.8 mM MgSO4, 0.4 mM KH2PO4, 0.3 mM Na2HPO4, 10 mM D-glucose, and 20 mM
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Na-HEPES; pH adjusted to 7.4). All the buffer salts were purchased from Sigma.
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Externally added serotonin solution to RN46A cells: RN46A cells are grown in coverslip
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bottomed Petri dishes (poly-L-lysine coated) and imaged for different fields. The cells are
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then exposed to the serotonin solution (400 µM) and same fields are imaged after 10 minutes
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of exposure. The control cells are treated with the TB buffer for 10 mins and subsequently
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imaged.
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Image analysis: The analysis is performed by selecting the soma of individual cells as
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regions of interest (ROI) and then computing the average brightness of individual vesicles in
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two
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http://rsbweb.nih.gov/ij/). The distribution of transmission to reflection (D(T)/D(R)) ratios are
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then calculated and plotted as a bar graph with their standard error of mean (SEM).
channels
using
image
J
(Open
source,
available
from
the
website:
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SUPPORTING INFORMATION
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Graph of fluorescence signal vs excitation power indicating three-photon excitation,
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serotonin fluorescence vs pH, and effect of SERT blocker on serotonin uptake.
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AUTHOR INFORMATION
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Current Addresses
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# Vienna University, Institute of Applied Physics-Biophysics Research, Getreidemarkt 9,
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1060, Vienna, Austria
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Corresponding Authors
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*E-mail:
[email protected] or
[email protected] 6
Author Contributions
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§
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U.T. and B.M. constructed the set-up; A.K.D., B.M., and U.T. performed the experiments;
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D.S. performed cell culture; S.M. conceptualized the experiment; A.K.D., U.T. and S.M. co-
A.K.D. and B.M. contributed equally to this work.
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wrote the manuscript.
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Notes:
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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We acknowledge the help of the TIFR Workshop in constructing the T-format PMT housing
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used here.
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REFERENCES [1] Canli, T., and Lesch, K. P. (2007) Long story short: the serotonin transporter in emotion regulation and social cognition, Nat Neurosci 10, 1103-1109. [2] Murphy, D. L., Andrews, A. M., Wichems, C. H., Li, Q., Tohda, M., and Greenberg, B. (1998) Brain serotonin neurotransmission: an overview and update with an emphasis on serotonin subsystem heterogeneity, multiple receptors, interactions with other neurotransmitter systems, and consequent implications for understanding the actions of serotonergic drugs, J Clin Psychiatry 59 Suppl 15, 4-12. [3] Pytliak, M., Vargova, V., Mechirova, V., and Felsoci, M. (2011) Serotonin receptors from molecular biology to clinical applications, Physiol Res 60, 15-25. [4] Das, A. K., Pandit, R., and Maiti, S. (2015) Effect of amyloids on the vesicular machinery: implications for somatic neurotransmission, Philos Trans R Soc Lond B Biol Sci 370. [5] Fuchs, E., and Flugge, G. (2004) Cellular consequences of stress and depression, Dialogues Clin Neurosci 6, 171-183. [6] Muller, C. P., and Homberg, J. R. (2015) The role of serotonin in drug use and addiction, Behav Brain Res 277, 146-192. [7] LeMarquand, D., Pihl, R. O., and Benkelfat, C. (1994) Serotonin and alcohol intake, abuse, and dependence: findings of animal studies, Biol Psychiatry 36, 395-421. [8] Kumar, M., Kaushalya, S. K., Gressens, P., Maiti, S., and Mani, S. (2009) Optimized derivation and functional characterization of 5-HT neurons from human embryonic stem cells, Stem Cells Dev 18, 615-627. [9] O'Connor, N., and Silver, R. B. (2013) Ratio Imaging: Practical Considerations for Measuring Intracellular Ca2+ and pH in Living Cells, Method Cell Biol 114, 387-406. [10] Balaji, J., Desai, R., Kaushalya, S. K., Eaton, M. J., and Maiti, S. (2005) Quantitative measurement of serotonin synthesis and sequestration in individual live neuronal cells, J Neurochem 95, 1217-1226. [11] Lee, M. H., Kim, J. S., and Sessler, J. L. (2015) Small molecule-based ratiometric fluorescence probes for cations, anions, and biomolecules, Chem Soc Rev 44, 41854191. [12] Maiti, S., Shear, J. B., Williams, R. M., Zipfel, W. R., and Webb, W. W. (1997) Measuring serotonin distribution in live cells with three-photon excitation, Science 275, 530-532. [13] Sarkar, B., Banerjee, A., Das, A. K., Nag, S., Kaushalya, S. K., Tripathy, U., Shameem, M., Shukla, S., and Maiti, S. (2014) Label-free dopamine imaging in live rat brain slices, ACS Chem Neurosci 5, 329-334. [14] Kaushalya, S. K., Nag, S., Ghosh, H., Arumugam, S., and Maiti, S. (2008) A highresolution large area serotonin map of a live rat brain section, Neuroreport 19, 717721. [15] Kaushalya, S. K., Desai, R., Arumugam, S., Ghosh, H., Balaji, J., and Maiti, S. (2008) Three-photon microscopy shows that somatic release can be a quantitatively significant component of serotonergic neurotransmission in the mammalian brain, J Neurosci Res 86, 3469-3480. [16] Sarkar, B., Das, A. K., Arumugam, S., Kaushalya, S. K., Bandyopadhyay, A., Balaji, J., and Maiti, S. (2012) The dynamics of somatic exocytosis in monoaminergic neurons, Front Physiol 3, 414. [17] Nag, S., Balaji, J., Madhu, P. K., and Maiti, S. (2008) Intermolecular association provides specific optical and NMR signatures for serotonin at intravesicular concentrations, Biophys J 94, 4145-4153. 8 ACS Paragon Plus Environment
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[18] Bruns, D., Riedel, D., Klingauf, J., and Jahn, R. (2000) Quantal release of serotonin, Neuron 28, 205-220. [19] Sulzer, D., Sonders, M. S., Poulsen, N. W., and Galli, A. (2005) Mechanisms of neurotransmitter release by amphetamines: a review, Prog Neurobiol 75, 406-433. [20] White, L. A., Eaton, M. J., Castro, M. C., Klose, K. J., Globus, M. Y., Shaw, G., and Whittemore, S. R. (1994) Distinct regulatory pathways control neurofilament expression and neurotransmitter synthesis in immortalized serotonergic neurons, J Neurosci 14, 6744-6753. [21] Balaji, J., Desai, R., and Maiti, S. (2004) Live cell ultraviolet microscopy: a comparison between two- and three-photon excitation, Microsc Res Tech 63, 67-71. [22] Lau, T., Proissl, V., Ziegler, J., and Schloss, P. (2015) Visualization of neurotransmitter uptake and release in serotonergic neurons, J Neurosci Methods 241, 10-17. [23] Gubernator, N. G., Zhang, H., Staal, R. G., Mosharov, E. V., Pereira, D. B., Yue, M., Balsanek, V., Vadola, P. A., Mukherjee, B., Edwards, R. H., Sulzer, D., and Sames, D. (2009) Fluorescent false neurotransmitters visualize dopamine release from individual presynaptic terminals, Science 324, 1441-1444. [24] Sames, D., Dunn, M., Karpowicz, R. J., Jr., and Sulzer, D. (2013) Visualizing neurotransmitter secretion at individual synapses, ACS Chem Neurosci 4, 648-651. [25] Pereira, D. B., Schmitz, Y., Meszaros, J., Merchant, P., Hu, G., Li, S., Henke, A., Lizardi-Ortiz, J. E., Karpowicz, R. J., Jr., Morgenstern, T. J., Sonders, M. S., Kanter, E., Rodriguez, P. C., Mosharov, E. V., Sames, D., and Sulzer, D. (2016) Fluorescent false neurotransmitter reveals functionally silent dopamine vesicle clusters in the striatum, Nat Neurosci 19, 578-586. [26] Rodriguez, P. C., Pereira, D. B., Borgkvist, A., Wong, M. Y., Barnard, C., Sonders, M. S., Zhang, H., Sames, D., and Sulzer, D. (2013) Fluorescent dopamine tracer resolves individual dopaminergic synapses and their activity in the brain, Proc Natl Acad Sci U S A 110, 870-875. [27] Mason, J. N., Farmer, H., Tomlinson, I. D., Schwartz, J. W., Savchenko, V., DeFelice, L. J., Rosenthal, S. J., and Blakely, R. D. (2005) Novel fluorescence-based approaches for the study of biogenic amine transporter localization, activity, and regulation, J Neurosci Methods 143, 3-25. [28] Alabi, A. A., and Tsien, R. W. (2012) Synaptic vesicle pools and dynamics, Cold Spring Harb Perspect Biol 4, a013680. [29] Rizzoli, S. O., and Betz, W. J. (2005) Synaptic vesicle pools, Nat Rev Neurosci 6, 57-69. [30] Hoopmann, P., Rizzoli, S. O., and Betz, W. J. (2012) Imaging synaptic vesicle recycling by staining and destaining vesicles with FM dyes, Cold Spring Harb Protoc 2012, 7783. [31] Gaffield, M. A., and Betz, W. J. (2006) Imaging synaptic vesicle exocytosis and endocytosis with FM dyes, Nat Protoc 1, 2916-2921. [32] Wersinger, C., Rusnak, M., and Sidhu, A. (2006) Modulation of the trafficking of the human serotonin transporter by human alpha-synuclein, Eur J Neurosci 24, 55-64. [33] Oaks, A. W., and Sidhu, A. (2011) Synuclein modulation of monoamine transporters, FEBS Lett 585, 1001-1006.
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Sample
LSMSS
M
fs Ti:Sapphire Laser
Objective
M λ /2 GLP L
Confocal Scanner
DM1
R
DM2
L
F
PC
L
L F
LSM Controller
T
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Figure 1. A Schematic representation of the optical setup for ratiometric imaging of serotonin (not to scale). Ti-Sapphire laser operating at 740 nm was steered into Zeiss LSM 710 confocal scanner. M: mirrors, λ/2: half wave plate, GLP: Glan laser polarizer, L: Lenses, F: Liquid CuSO4 Filter, LSMSS: Laser scanning microscope scanning stage, DM1: 670 nm short pass dichroic mirror, DM2: 360 nm long pass dichroic mirror, R,T: photomultiplier tube detectors, PC: computer.
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Figure 2. Three-photon fluorescence intensity ratio (T/R) obtained from serotonin solution as a function of concentration. (Inset) Area-normalized fluorescence emission spectra of serotonin at different concentrations (at 60 µM, 1 mM, 12.5 mM , 25 mM, 50 mM, 100 mM, 200 mM , 300 mM , 400 mM , 500 mM and 600 mM respectively). The iso-emissive point at 376 nm is shown with a dotted vertical line. Solid red line schematically shows the spectral characteristic of the UV dichroic (360 nm long pass). The reflected signal is collected by detector R (green box), while the transmitted signal is collected by detector T (blue box). Inset adapted from Nag et al.17 The arrow indicates the direction of change of signal with increase of concentration.
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Figure 3. The effect of externally added serotonin on vesicular content in RN46A cells. (I) Three-photon excitation images of serotonin in RN46A cells at (a) 0 min and (b) 10 min after vehicle treatment in one of the channels. Images of serotonin in RN46A cells at (c) 0 min and (d) 10 min after treating the same cells with externally added serotonin solution (400 µM) (image intensities are false color coded). (II) Histogram of the mean ratios of signals collected in the transmission and reflection channels (T/R) at 0 min (black) and 10 min (red) after vehicle treatment. (III) Histogram of the mean T/R ratio 0 min (black) and 10 min (red) after treatment with externally added serotonin solution (400 µM) (error bar = standard error of the mean). Scale bars = 10µm.
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5-HT cells
Page 13 ofACS 13 Chemical Neuroscience Objective 740 nm 7540 3-photon excitation of 5-HT
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670 nm Dichroic
Reflection Channel
360 nm Dichroic
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Transmission Channel 5-HT conc in vesicles
