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Letter
Fluorogenic Detection of Monoamine Neurotransmitters in Live CellsKallol Bera, Anand Kant Das, Ananya Rakshit, Bidyut
Sarkar, Anoop Rawat, Barun Kumar Maity, and Sudipta MaitiACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00391 • Publication Date (Web): 11 Dec 2017
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1
Fluorogenic Detection of Monoamine 1
Neurotransmitters in Live Cells 2
Kallol Bera, Anand Kant Das, Ananya Rakshit, Bidyut Sarkar, Anoop Rawat, Barun Kumar 3
Maity, Sudipta Maiti* 4
Department of Chemical Science, Tata Institute of Fundamental Research, Homi Bhabha Road, 5
Colaba, Mumbai 400005, India 6
*e-mail: [email protected] 7
8
9
ABSTRACT: 10
Monoamine neurotransmission is key to neuromodulation, but imaging monoamines in live 11
neurons has remained a challenge. Here we show that externally added ortho-phthalaldehyde 12
(OPA) can permeate live cells and form bright fluorogenic adducts with intracellular 13
monoamines (e.g. setrotonin, dopamine and nor-epinephrine) and with L-DOPA, which can be 14
imaged sensitively using conventional single-photon excitation in a fluorescence microscope. 15
The peak excitation and emission wavelengths (λex = 401 nm and λem = 490 nm for serotonin; λex 16
= 446 nm and λem = 557 nm for dopamine; and λex = 446 nm and λem = 544 nm for nor-17
epinephrine) are accessible to most modern confocal imaging instruments. The identity of 18
monoamine containing structures (possibly neurotransmitter vesicles) in serotonergic RN46A 19
cells is established by quasi-simultaneous imaging of serotonin using three-photon excitation 20
microscopy. Mass spectrometry of cell extracts and of in vitro solutions helps us identify the 21
chemical nature of the adducts, and establishes the reaction mechanisms. Our method has low 22
toxicity, high selectivity and the ability to directly report the location and concentration of 23
monoamines in live cells. 24
25
KEYWORDS: serotonin imaging, dopamine imaging, norepinephrine imaging, monoamine 26
microscopy, Falck Hillarp method 27
28
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1
2
Monoaminergic neurotransmission is important for processes related to mood, memory, reward 3
and addiction, and in the context of diseases such as Parkinson’s, Alzheimer’s and depression 1-5
. 4
Serotonin (5-HT), dopamine (DA) and norepinephrine (NE) are major monoamine 5
neurotransmitters (MNT) in the brain, and the associated neuronal circuits are targets of many 6
common pharmaceutical agents and drugs of abuse6-8
. Monoamines are packed at a concentration 7
of hundreds of mM in the neurotransmitter vesicles and to a level of tens of mM in some of the 8
non-neuronal vesicles9, 10
. Various strategies have been devised to detect and image these 9
neurotransmitters in biological tissues with subcellular resolution. Microscopic imaging has been 10
achieved with immunohistochemistry, but the cells have to be fixed7. Direct detection of 5-HT 11
and DA with ultraviolet radiation has been attempted in live cells, but the UV radiation severely 12
damages the cells, and available UV optics are of lower quality than visible wavelength optics11,
13
12. We and others have shown that three photon excitation can be effectively used to image 14
serotonin in live neurons in the near-infra red by harnessing its intrinsic mid-ultraviolet 15
autofluorescence13-16
. Recently, we have also imaged intracellular DA with sub-µm three-16
dimensional resolution using two photon excitation9, and demonstrated label-free ratiometric 17
imaging of serotonin using three-photon excitation17
. However, these techniques require a 18
modified confocal microscope and pulsed femtosecond lasers. A detection method based on 19
conventional (one-photon) excitation of fluorescence would be a boon to the community due to 20
its accessibility and convenience. 21
Conventionally excited Fluorescent false neurotransmitters (FFNs) are selectively transported 22
into vesicles containing monoamine transporters, and allow visualization of their exocytosis from 23
individual presynaptic terminals18-20
. However, FFNs are loaded into all secretory vesicles 24
expressing VMAT (vesicular monoamine transporter) without discrimination to cell type, and 25
therefore this approach cannot distinguish distinct cell populations that secrete a particular 26
neurotransmitter21
. Also, FFN fluorescence does not report the concentration of native 27
neurotransmitters at any given location. In recent years, a number of cortical amine sensors have 28
been reported, including fluorescent ribonucleopeptide (RNP) complexes22
, and boronic acid 29
based fluorescent compounds23, 24
. However, none of these methods represents a practical 30
approach for in vitro and in vivo cellular analysis and imaging25, 26
. It is thus imperative to 31
explore a direct but benign method for their quantitative detection and visualization in live 32
samples. 33
Since one-photon excitation (1-PE) in the visible cannot directly excite monoamines, we have 34
to rely on the availability of fluorogenic reactants. Our method is inspired by the work of Falck 35
and Hillarp, who discovered that exposing dry brain tissue to formaldehyde vapor (coupled with 36
an oxidation step) imparts a bluish green fluorescence to monoaminergic cells27, 28
. This enabled 37
great progress in the field of monoaminergic neurotransmission, but the experimental 38
requirements precluded the detection of monoamines in live cells29
. 39
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Here, we have chosen a small organic molecule ortho-phthalaldehyde (OPA) to image and 1
quantify native neurotransmitters in live neuronal cells30-32
. We hypothesized that the highly 2
nucleophilic primary amine group of MNT would readily form an isoindole with OPA, which 3
will be further stabilized by the aromatic ring of the individual neurotransmitters, e.g. catechol 4
amine (for DA) and indole (for 5-HT). The suggested reaction schemes are shown in Scheme 1. 5
DA can give rise to compounds I, II and III, while 5-HT can yield IV, V and VI. The chemical 6
reaction is related to that of formaldehyde in the Falck and Hillarp method, but we hypothesized 7
that the extra phenyl ring would make the fluorescence more red-shifted, and might help the 8
compound penetrate cell membranes better. The lower volatility of this compound, compared to 9
that of formaldehyde, also makes it much easier and less toxic to handle27, 28
. In addition, the 10
extreme toxicity of formaldehyde does not permit its use in live cells, and this modification may 11
make it less toxic to cells. We note that OPA has already been used for labeling histamine in 12
plant tissue33
. 13
We tested our hypothesis in vitro by adding 500 µL of 100 µM aqueous MNT (DA or 5-HT) 14
solution (colorless) to 500 µL of 100 µM aqueous OPA solution (colorless) separately. The 15
solutions first turned violet and then changed to light green and ultimately to dark green, 16
indicating the formation of MNT-OPA adduct. Mass spectrometry with a MALDI-TOF mass 17
spectrometer revealed the formation of an isoindole ring [observed m/z for DA = 250.95, 368.33, 18
500.67, 619.94 (Fig. S1-7); m/z for 5-HT = 272.01, 274.35, 276.03, 293.11(Fig. S8-11)], and 19
supported the proposed reaction scheme. For DA, we did not get the signature of the product III 20
in the MALDI spectrum. However, we have found higher molecular mass which is possibly due 21
to polymerization of DA-OPA adducts through catechol moiety. These polymerization reactions 22
are described in the Fig. S3-S6. ESI-MS also supports the formation of adduct I (Fig. S7). For 5-23
HT, due to the absence of such reactive moiety, no polymerization was observed. ESI-MS 24
further support the formation of product V and VI (Fig. S10, S11). 25
We then tested the fluorescence properties of the coloured products. The DA–OPA adduct has 26
its excitation maximum (λex) at 446 nm (Fig. 1A, magenta) and it emits in the region from 500-27
640 nm, with a peak at 557 nm (Fig. 1B, magenta). 5HT-OPA has the excitation maxima at 401 28
nm (Fig. 1A, red) and emits in the region of 470-620 nm, with a peak at 490 nm (Fig. 1B, red). 29
Excitation and emission spectra of the OPA adducts of nor-epinephrine and L-DOPA are similar 30
to that of DA-OPA (Fig. 1A and 1B, green and blue respectively). The reaction of MNT with 31
OPA at pH 5.5 (estimated intravesicular pH) is nearly as efficient as in neutral pH 7.4 (Fig. 1C). 32
OPA by itself has very little fluorescence (Fig. 1B, black), so the fluorescence enhancement for 33
DA-OPA compared to OPA is > 20X (Fig. 2). A small peak at 532 nm is visible in the emission 34
spectra. This is an artefact due to Raman scattering. We then tested the selectivity of this 35
fluorogenic reaction against other common cellular components. The MNTs are derived from 36
aromatic amino acids, so we investigated the selectivity of the sensor against the 20 common 37
amino acids (listed in the legend of Fig. 2) at the same concentration. We also tested 17 other 38
compounds using the same protocol (Fig. 2). The fluorescence intensity of no other compound 39
exceeded 10% of the norepinephrine value. Epinephrine (EN) cannot form a fluorescence adduct 40
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similar to norepinephrine due to its secondary amine functional group. We note that the 1
intravesicular neurotransmitters concentration is >100 mM14
, so this level of enhancement by the 2
other compounds would be expected to provide very low background fluorescence, resulting in 3
very high selectivity for the neurotransmitters. The distinct emission spectra also provide some 4
specificity amongst the well-detected compounds (5-HT, DA, NE and L-DOPA). Since 5
intravesicular pH is ~ 5.5, we hypothesize that OPA can be used for intravesicular monoamine 6
neurotransmitter detection in live cells. 7
8
Next, we attempted fluorogenic detection of 5-HT and DA in live cells, using the cell lines 9
MN9D (dopaminergic neuronal cell line), RN46A (serotonergic neuronal cell line) and 10
HEK293T (somatic cell line, used as a negative control). The cells were cultured according to 11
protocols described elsewhere9. They were exposed to 100 µM OPA for 30 min at 37 °C, washed 12
with Thomson’s buffer to remove the remaining OPA, and imaged in a confocal microscope 13
(Zeiss LSM710) using single photon excitation, with λex = 488 nm. Fluorescence microscopic 14
images showed that OPA penetrates cell membranes and reacts with MNT to form fluorescent 15
products (Fig. 3). The enhancement of fluorescence was clear both for MN9D cells (compare 16
Fig. 3a and 3c), and for RN46A cells (Fig. 3d and 3f). However, HEK293T cells show minimal 17
increase of fluorescence (Fig. 3g and 3i). This is consistent with our expectation since HEK293T 18
cells are not expected to contain monoaminergic vesicles. RN46A cells showed perinuclear 19
luminescence in the cytoplasm, a pattern similar to that observed by us earlier using multiphoton 20
microscopy9, 29, 34
. However, MN9D cells showed a more diffuse cytoplasmic fluorescence in 21
(Fig. 3c). This was also similar to the results obtained by us earlier using direct multiphoton 22
imaging of dopamine9. To further verify whether the fluorescence indeed arises from the 23
neurotransmitters, we probed a set of RN46A cells with direct three-photon excitation (3-PE) of 24
serotonin at 740 nm (before OPA treatment, Fig. 3j), and subsequently by single photon 25
excitation (1-PE) at 488 nm, after treating them with OPA (Fig. 3k). A Ti:Sapphire (MIRA, 26
Coherent Inc., USA) laser operating at 740 nm and producing ~ 100 fs pulses (repetition rate, 76 27
MHz) was used for three photon excitation, and the cells were visualized using a custom-28
modified confocal microscope (LSM710, Zeiss, Germany). The radiation was separated from the 29
serotonin fluorescence by a dichroic mirror (670dcxruv, Chroma, USA), and detected in the non-30
descanned part of the path.17
The signal was passed through a 1-cm thick liquid CuSO4 filter to 31
block the excitation light. The filtered signal was detected by an analog photomultiplier tube 32
(Model: P30A-01 Electron Tubes Limited, UK)28, 29
, which was externally connected to a custom 33
signal input channel of the LSM 710 confocal microscope. 34
The images obtained from the 3-PE and 1-PE experiments showed very similar punctate 35
objects, which may be vesicles or vesicular clusters. While there was some movement of these 36
objects and the morphology of the cells did change somewhat in the intervening time, the 37
similarities between the structures are obvious. 38
To further confirm that the same chemical reactions occurred inside the cells as had been 39
observed in vitro, we performed mass spectroscopy of the cell extracts after OPA treatment. 40
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RN46A cells were incubated with 100 µM OPA in Thomson’s buffer for 30 min and then lysed 1
by vigorous sonication. The lysate was collected for mass spectrometry. Though the small 2
amounts obtained compromised the signal-to-noise ratio, the m/z peaks at 277.44 (VI+H+), 3
297.51 (IV+Na+), 313.0 (IV+K
+) and 331.82 (V+K
+) were observed in the OPA-treated cells 4
(Fig. S12). The results are therefore consistent with the interpretation that the same products 5
were formed inside the cell. 6
We further probed the applicability of our method by measuring the potential toxic effects of 7
OPA in live cells. RN46A cells were plated on 96-well plates and were treated with various 8
concentrations [10, 50, 100, 500, 1000 µM] of OPA for 24 hours. Cell viability was measured 9
with MTT assay35
. Cells were grown in 96-well plates and test compounds (10, 50, 100, 500, 10
1000 µM of OPA) were added to make a final volume of 100 µl/well and incubated according to 11
the respective treatment time-points. 100 µl MTT solution (>98% purchased from SigmaAldrich) 12
was added to each well to achieve a final concentrate ion of 0.5 mg/ml. The plate was incubated 13
for 4 hours at 37°C. 100 µl of the solubilizing solution (50% (v/v) dimethylformamide (DMF) in 14
2% (v/v) glacial acetic acid, 20% (w/v) sodium dodecyl sulfate (SDS) was then added to each 15
well to dissolve the formazan crystals. In order to ensure complete solubilization the plate was 16
left in the 37°C incubator overnight. The absorbance was recorded at 570 nm on the following 17
day. The results shown in Fig. 4 are the values of absorbance measurement (these values are not 18
normalized), which provide a measure of the toxicity of the different concentrations of OPA. We 19
found that OPA was not significantly toxic at 100 µM (which was the concentration used for the 20
cell imaging studies) and only moderately toxic at 500 µM. 21
In conclusion, our results demonstrate that fluorogenic reactions with OPA can provide an 22
accessible fluorescence imaging approach for monoamine neurotransmitters in live neuronal 23
cells. 24
25
26
ASSOCIATED CONTENT 27
28
Reaction Scheme for Dopamine and OPA adduct formation, Mass Spectra of the Dopamine OPA 29
adduct, MALDI spectra of the Dopamine-OPA adduct, Reaction mechanism for OPA dopamine 30
adducts for product I and II, Plausible Reaction Mechanism for Higher Molecular Weight 31
Adduct Formation, ESI-MS spectra of the Dopamin OPA adduct, OPA Serotonin Reaction, Mass 32
spectra of the Serotonin OPA adduct, Fluorescence Spectroscopy of Dopamine-OPA and 5-33
HT:OPA adduct, MN9D, RN46A and HEK cell culture, confocal imaging studies of cells, 34
Colocalization experiments (Multiphoton imaging followed by single photon), Mass 35
Spectrometry with OPA treated RN46A cells. 36
37
AUTHOR INFORMATION 38
39
Corresponding Author 40
41
*Email: [email protected] 42
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1
ORCID 2
Sudipta Maiti: 0000-0002-6540-7472 3
4
Present address: K.B., Division of Biology and Biological Engineering, California Institute of 5
Technology, Pasadena, CA 91125 6
A.K.D., Vienna University of Technology, Institute for Applied Physics - Biophysics group, 7
Getreidemarkt 9, 1060 Vienna, Austria 8
B.S., Molecular Spectroscopy Laboratory, RIKEN, 2-1, Hirosawa, Wako351-0198, Japan 9
A.R., University of Southern California, Los Angeles 90033, California 10
11
12
13
Notes 14
15
The authors declare no competing financial interest. 16
17
18
ACKNOWLEDGEMENTS 19
20
We thank Dr. Bappaditya Chandra (TIFR) and Prof. Soumen Basak (SINP) for their help and 21
support. 22
23
24
25
26
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Scheme 1: Formation of possible products in a reaction between monoamine 3
neurotransmitters (dopamine and serotonin) and OPA. 4
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Figure 1: (A) and (B) represent excitation and emission spectra respectively of 10 µM OPA upon 4
addition of 10 µM of each analyte in pH 7.4 PBS buffer. (OPA: Black, Serotonin + OPA: red, L-5
Dopa + OPA: blue, Dopamine + OPA: magenta, Nor-epinephrine + OPA: green). For recording 6
excitation spectra emission was monitored at 560 nm. For the emission spectra, the excitation 7
wavelengths for OPA, serotonin, L-dopa, dopamine and nor-epinephrine are at 450 nm, 400 nm, 8
440 nm, 450 nm and 450 nm respectively. In (C), black and red traces represent normalized 9
emission spectra of 100 µM serotonin + 100 µM OPA at pH 7.4 and 5.5 respectively, while blue 10
and magenta represent100 µM dopamine + 100 µM OPA at pH 7.4 and 5.5 respectively. 11
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414039383736353433323129 3028272625242322212012
Fl. Int.@ 560 nm
1 2 3 4 5 6 7 8 9 10 11 13 14 15 16 17 18 19 2
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Figure 2: Fluorescence intensity (λex = 450 nm, λem = 560 nm, unless mentioned otherwise) of 5
10 µM OPA upon addition of 10 µM of each analyte in pH 7.4 PBS buffer (λex = 450 nm). 1: 6
Ascorbic acid; 2: Adenosine monophosphate; 3: Arginine; 4: BSA; 5: Citric acid ; 6: Cysteine; 7: 7
Glucose; 8: Glutamic acid; 9: Glycine; 10: Guanidine; 11: Guanosine; 12: Homo-cysteine 8
thiolactone; 13: Histidine; 14: HSA; 15: Imidazole; 16: Isoleucine; 17: Lipoic acid; 18: Lysine; 9
19: Cysteine + Lysine; 20: Methionine; 21: NADPH; 22: Phenylalanine; 23: Piperonyleamine; 10
24: Serine; 25: Thiamine; 26: Thymine; 27: Tryptophanamide; 28: Tryptophan; 29: Tyrosine; 30: 11
OPA; 31: PCA; 32: Epinephrine; 33: Glutamine; 34: Na+; 35: Mg
2+; 36: K
+; 37: Ca
2+; 38: 12
Serotonin (λem = 490 nm); 39: L-Dopa; 40: Dopamine; 41: Nor-epinephrine. 13
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Figure 3: (a – c) : MN9D cells incubated with 100 µM OPA (a) 0 min after incubation, (b)
transmission image, (c) 30 min after incubation. (d-f) RN46A incubated with 100 µM OPA (d) 0 min
after incubation, (e) transmission image, (f) 30 min after incubation. (g-i) HEK (Control) cells
incubated with 100 µM OPA. (g) 0 min after incubation, (h) transmission image, (i) 30 min after
incubation. (j – k) Comparison of observed serotonin containing structures in RN46A cells. (j)
multiphoton image (λex = 740 nm) (k) single photon confocal image (λex = 488 nm), after 100 µM OPA
treatment. Scale bar is 20 µm (for a-i), 10 µm (for j-k).
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Figure 4: Toxicity of different concentrations of OPA measured for RN46A cells using MTT 22
assay. 23
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0.0
0.5
1.0
1.5
2.0
2.5
10 µΜ
ΟPA
50 µΜ
ΟPA
100 µΜ
ΟPA
500 µΜ
ΟPA
1000 µΜ
ΟPA
Absorbance
Sham
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