Specific Fluorescent Probe Based on "protect-Deprotect" to Visualize the Norepinephrine Signaling Pathway and Drug Intervention Tracers

Article abstract of DOI:10.1021/jacs.0c08956

In recent years, increased social pressure and other factors have led to a surge in the number of people suffering from depression: studies show that quite a few people will experience major depression in their lifetime. Currently, it is widely believed t

Full text of DOI:10.1021/jacs.0c08956

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Specific Fluorescent Probe Based on “Protect−Deprotect” To
Visualize the Norepinephrine Signaling Pathway and Drug
Intervention Tracers
Na Zhou, Fangjun Huo, Yongkang Yue, and Caixia Yin*
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ABSTRACT: In recent years, increased social pressure and other factors have led to
a surge in the number of people suffering from depression: studies show that quite a
few people will experience major depression in their lifetime. Currently, it is widely
believed that the internal cause of major depression is reduced levels of
norepinephrine (NE) in brain tissue. Norepinephrine is very similar in structure
and chemical properties to the other two catecholamine neurotransmitters,
epinephrine (EP) and dopamine (DA). These three neurotransmitters are
synthesized sequentially through enzymatic reactions in the biological system.
Therefore, design of a norepinephrine-specific fluorescent probe is very challenging.
In this work, we utilized a “protect−deprotect” strategy: longer emission wavelength
cyanine containing water-soluble sulfonate was protected by a carbonic ester linking
departing group thiophenol; the β-hydroxy ethyl amine moiety of norepinephrine
may react with the carbonic ester via nucleophilic substitution and intramolecular
nucleophilic cyclization to release the fluorophore. The process realized the specific
red fluorescence detection of norepinephrine. Imaging of the norepinephrine nerve signal transduction stimulated by potassium ion
was studied. More importantly, real-time fluorescence imaging of norepinephrine levels in the brain of rats stimulated by
antidepressant drugs was studied for the first time.
extremely difficult task to design fluorescent probes that can
respond specifically to norepinephrine.
INTRODUCTION
■
The signaling mode between neurons is mainly dependent on
the secretion, transmission, and uptake of neurotransmitters.
Neurotransmitters are chemicals released by neurons that bind
to specific receptors in specific ways. Interactions between
neurotransmitters and receptors are usually transient, lasting
from milliseconds to minutes. However, its effects can make
long-term changes in the target cell lasting for hours or days,
which can have a huge impact on the body.1 In recent years,
with the increase of social pressure, the number of depressive
symptomatology patients has soared. According to the World
Health Organization, the number of people suffering from
depression globally has exceeded 264 million. Depression has
become the leading cause of disability worldwide and a major
contributor to the global burden of disease.2 Studies have
shown that depression is closely related to decreased levels of
norepinephrine (NE) in the brain.3 Norepinephrine, as the
central substance in the synthesis of monoamine catechol-
amine neurotransmitters, is produced by the enzymatic
reaction of dopamine (DA) and then epinephrine (EP)
under the action of phenylethanolamine N-methyltransferase
(PMNT).4,5 It can be seen that the structures and properties of
the three monoamine neurotransmitters are similar and
synthesized successively through enzymatic reactions. It is an
Traditional methods for the detection of neurotransmitters
mainly rely on electrochemical analysis and mass spectrometry,
However, they often have some limitations in situ in vivo.6−9
At present, the fluorescence method for the detection and
marking of norepinephrine mainly included the following
aspects. The Kleinfeld team reported the cell-based neuro-
transmitter fluorescently engineered reporters; CNiFERs was
used for fluorescence imaging of changes in NE or DA
extracellular concentration during nerve signal transduction.
The system indirectly detected the release of neurotransmitters
using Ca2+ fluorescent probes to detect the concentration of
intracellular Ca2+ during exocytosis.10 Sames reported imaging
the release of NE during nerve signal transduction using a
pseudoneurotransmitter fluorescent dye FFN270 specifically
labeled with norepinephrine cells.11 Li reported that the
specific binding of NE and GPCR was used to regulate the
Received: August 19, 2020
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deprotonation process of cpEGFP connected with GPCR, and
then an enhanced single-channel fluorescence signal was
released to realize specific, highly sensitive, and real-time
monitoring of NE.12 The Glass group research has long been
concentrated on using small organic molecules for the
fluorescence detection of neurotransmitters using the aldehyde
group conjugated with the fluorophore as the reaction site,
which has different bonding constants with the primary amine
of targets resulting in different fluorescence signal changes.13,14
Therefore, detection of the neurotransmitter content and
release markers by fluorescent probes based on a norepinephr-
ine-specific response is still a challenge. In our previous study,
we initially found that the unique 2-aminoethanol moiety of
norepinephrine had a cascade nucleophilic substitution with
carbonates to release the fluorophore, thus realizing the
specific detection of norepinephrine. Moreover, it was
successfully applied to the specific labeling of norepinephrine
in brain tissue slices through dual immunofluorescent probes.15
However, the water solubility and short emission wavelength
of the probe limited its further application.
194.04537 [M − H]− were found in the HR-MS (Figure
S3). Subsequently, the UV−vis and fluorescence response of
the probe to norepinephrine in vitro was then carried out.
Since the water solubility of the probe was greatly improved,
we carried out spectrometric determination in the PB system
(pH 5.0). Five millimolar NE was added to 2 mL of PB (pH
5.0) containing a 10 μM probe. As shown in Figure 1a, the UV
In this work, we realized red fluorescence detection for
norepinephrine in an aqueous solution using cyanine as a
fluorophore to lengthen its emission wavelength and introduce
sulfonate to greatly improve its water solubility (Scheme 1).
Scheme 1. Response Mechanism of the Probe with
Norepinephrine
Figure 1. (a) UV−vis response of a 10 μM probe upon 5 mM NE in
PB (pH 5.0) for 0−120 min. (b) Fluorescent response of a 10 μM
probe upon 5 mM NE in PB (pH 5.0) for 0−60 min (λex = 550 nm,
slit = 10 nm/5 nm). (c) Time dependence on a 10 μM probe upon 5
mM NE, DA, and EP for 0−60 min. (d) Selectivity of a 10 μM probe
upon 5 mM neurotransmitters (DA, EP, NE, 5-HT, GABA) and
GSH, 500 μM amino acids (lys, thr, ser), 100 μM Cys, and 10 μM
Hcy.
absorption of the system at 675 and 775 nm gradually
decreased over time, while that at 550 nm gradually increased.
Accordingly, after addition of NE, the fluorescence intensity of
the system gradually increased at 640 nm, which was 14 times
higher than that of the system containing only the probe
(Figure 1b). The spectral properties of the system after
reaction were in good agreement with those of the cyanine
ketone itself (Figure S4).
Subsequently, the probe successfully labeled norepinephrine-
rich vesicles in PC12 cells and imaged the process of
norepinephrine exocytosis when stimulated by high potassium.
For the first time, in situ imaging of norepinephrine elevation
in vivo induced by fluoxetine (a typical antidepressant) has
been achieved. It will have certain practical significance for the
research and screening of depression drug therapy.
In further experiments, as we designed, both dopamine and
epinephrine still induced less fluorescent intensities enhance-
ment compared with norepinephrine (Figures 1c). Besides we
also studied other neurotransmitters (5 mM), such as 5-HT,
GABA, and amino acids (500 μM), such as threonine, serine,
and lysine. Studies have shown that none of them induced a
distinct fluorescent change of the probe in the detection
system (Figures 1d and S5). We also investigated 100 μM
cysteine/10 μM homocysteine with a similar mercaptoethyl-
amine moiety and 5 mM GSH, which all did not induce a
significant fluorescent response (the cellular concentration of
unbounded Cys/Hcy is nnanomolar to micromolar, Figure
1d). The probe response time in this work was greatly
shortened, and the reaction was completed within 50 min so as
to minimize the loss of norepinephrine caused by REDOX.
Imaging of Endogenous Norepinephrine in Living
Cells. As a neurotransmitter, norepinephrine is usually
concentrated in specialized cells or tissues. We selected several
kinds of cells to mark endogenous norepinephrine. When the
20 μM probe was cultured with Hela cells and HepG-2 cells for
40 min at 37 °C, no fluorescence emission was observed in the
RESULTS AND DISCUSSION
■
Synthesis and Characterization. Compounds were
synthesized according to the procedure in Scheme S1 in the
Supporting Information. Eventually the probe was obtained as
a dark green solid (0.18 g, 21%). 1H NMR (600 MHz,
CD3OD) δ 7.85 (d, J = 14.0 Hz, 2H), 7.52 (d, J = 7.3 Hz, 2H),
7.48 (d, J = 7.7 Hz, 2H), 7.39 (t, J = 7.6 Hz, 2H), 7.34 (d, J =
7.9 Hz, 2H), 7.30 (d, J = 7.6 Hz, 2H), 7.25 (t, J = 7.3 Hz, 2H),
6.25 (d, J = 14.1 Hz, 2H), 4.17 (t, J = 6.7 Hz, 4H), 2.85 (t, J =
6.7 Hz, 4H), 2.68 (s, 4H), 2.35 (s, 3H), 1.99−1.88 (m, 10H),
1.73 (s, 12H); 13C NMR (151 MHz, CD3OD) δ 144.1, 104.5,
54.3, 51.9, 51.8, 51.6, 51.5, 51.3, 51.2, 51.1, 47.5, 31.1, 29.8,
24.6 (Figure S1). HR-MS: calcd 857.29696; found 857.29698
(Figure S2).
Norepinephrine Detection Using a Probe. The specific
response mechanism of the probe to norepinephrine was
confirmed again by mass spectrometry. In the final reaction
products both the cyanine ketone peak at 353.13792 [Cy7
-OH]2− and the five-membered ring compound peak
B
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red channel (Figure 2). PC12 cells are often used to build
neural models because of its characteristics of nerve cells and
Figure 2. PC12 cells, Hela cells, and HepG2 cells with the probe (20
μM) cultured in the red channel (5−40 min) (λex: = 561 nm, bar = 20
μm).
secretory cells. Studies have shown that PC12 cells can secrete
norepinephrine-rich vesicles and have exocytosis.16 Surely,
after the probe was cultured with PC12 cells in similar
conditions, an obvious red fluorescence emission could be
observed and it increased over time (Figure 2). The results
showed that the probe could mark the norepinephrine in the
vesicle.17
Figure 3. (a−e) Probe-loaded PC12 cells with high K+ (100 μL) in
the red channel (0−40 s). (f−j) Probe-loaded PC12 cells with PBS in
the red channel (0−40 s). (k) Time dependence on probe-loaded
PC12 cells with high K+ (100 μL) and PBS in 0−40 s (λex = 561 nm,
bar = 20 μm).
Dopamine β-Hydroxylase (1:1000, MAB308; Millipore) for 2
h followed by a secondary antibody cocktail of Goat Anti-
Rabbit IgG/Cy3 (1:150, bs-0295G-Cy3; Bioss Antibodies) and
Goat Anti-Mouse IgG/ALexa Fluor 350 (1:100, bs-0296G-
AF350; BiossAntibodies) for 30 min, which were then stained
with the probe (10 μM) for 120 min. As shown in Figure 4, the
Imaging of the Process of Norepinephrine Exocy-
tosis. The study of signal transduction of neurotransmitters is
beneficial to the prevention and intervention of neurological
diseases. It is reported that the action potential produced by
high concentrations of potassium stimulating the cell causes
voltage-dependent calcium-ion channel depolarization to open
the calcium-ion channels. Driven by the electrochemical
potential, calcium ions flow into cells from the outside; the
increased concentration of calcium in the cytoplasm triggers
fusion of vesicles with the cytoplasmic membrane. Con-
sequently, it stimulates the release of neurotransmitters.18 In
this work, PC12 cells loaded with probes-labeled norepinephr-
ine vesicles were stimulated with high-K+ solution, and the
intensity of the fluorescent spots in the cells gradually
decreased. However, under the same conditions, the cells
were stimulated with PBS (without potassium ion). The
fluorescent intensity was almost unchanged in the red channel
(as shown in Figure 3). The results indicated that increased K+
concentration led to cell exocytosis and that the probe realized
visualization of the norepinephrine signaling pathway.
Noradrenaline Visualization in Brain Tissue. As a long-
wavelength red emission fluorescent probe, we used it for brain
tissue labeling. Noradrenergic neurons are distributed in the
brainstem nucleus locus coeruleus. Dual immunolabeling for
TH and DBH or DBH and PNMT is a successful and effective
method. In this work, we still employed a primary antibody
cocktail of Rabbit Anti-Tyrosine Hydroxylase Polyclonal
antibody (1:1000, bs-0016R; Bioss Antibodies) and Anti-
Dopamine β-Hydroxylase (1:1000, MAB308; Millipore) for
TH and DBH dual-labeling experiments for 2 h. This was
followed by a secondary antibody cocktail of Goat Anti-Rabbit
IgG/Cy3 (1:250, bs-0295G-Cy3; Bioss Antibodies) and Goat
Anti-Mouse IgG/ALexa Fluor 350 (1:100, bs-0296G-AF350;
Bioss Antibodies) for 30 min, which were then stained with the
probe (10 μM) for 120 min. For PNMT and DBH dual-
labeling experiments, the sections were processed with a
primary antibody cocktail of Rabbit Anti-PNMT Polyclonal
antibody (1:1000, bs-3912R; Bioss Antibodies) and Anti-
Figure 4. (a1, a2) Immunofluorescence the Cy3-labeled TH-positive
region. (b1, b2) Probe-labeled brain tissue. (c1, c2) Immunofluor-
escence AF350-labeled DBH-positive region. (d1) Merged image of
a1 and b1. (d2) Merged image of a2 and b2. (e1) Merged image of b1
and c1. (e2) Merged image of b2 and c2 (Cy3: λex = 548 nm. AF350:
λex = 405 nm. Probe: λex = 561 nm. Bar = 200 μm).
area involving the probe (b1, b2) and DBH-AF350 (c1,c2)
fluorescent emission showed extensive overlap (e1, e2), which
indicated that the probe has a specific labeling ability for
noradrenergic neurons. Also, it still showed the probe
specifically labeled norepinephrine over epinephrine by dual
immunolabeling for DBH and PNMT (Figure 5). This
bidirectional detection strategy strongly proved that the
probe can efficiently and specifically label norepinephrine in
the tissue level.
Fluorescent Imaging In Vivo. The penetration depth of
imaging is always limited by the wavelength due to the long
wavelength of the probe effectively supplementing some of the
limitations of fluorescent imaging applications; we carried out
fluorescent in vivo imaging. The probe (20 μM) was injected
on the right side of the back of mice, and then norepinephrine
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Figure 7. (a−c) In vivo imaging of rats intraperitoneally injected with
saline. (d−f) In vivo imaging of rats intraperitoneally injected with
fluoxetine (10 mg/kg). (g) Fluorescent intensity of ROIs normal
saline and fluoxetine injected rats (λex = 561 nm).
Figure 5. (a1, a2) Immunofluorescence the Cy3-labeled PNMT-
positive region. (b1, b2) Probe-labeled brain tissue. (c1, c2)
Immunofluorescence AF350-labeled DBH-positive region. (d1)
Merged image of a1 and b1. (d2) Merged image of a2 and b2. (e1)
Merged image of b1 and c1. (e2) Merged image of b2 and c2 (Cy3:
λex = 548 nm. AF350: λex = 405 nm. Probe: λex = 561 nm. Bar = 200
μm).
group produces the intramolecular PET effect resulting in no
fluorescence emission. Subsequently, using the unique amino
ethanol structural unit of NE, through cascade nucleophilic
and leaving reactions, deprotection and release of the
fluorophore occurs. Using the fluorescent probe with a long-
wavelength red emission, we verified the specific response of
the probe to NE in a complex biological environment through
cell imaging, slice imaging, and in vivo imaging. In this work,
we applied the probe for fluorescence imaging of norepinephr-
ine exocytosis signaling pathways under high K+ stimulation.
The first visualization of the effect of antidepressant drugs on
norepinephrine intervention was achieved.
(200 μM) was injected in the same area. The probe (20 μM)
and PBS (same volume with NE) were injected on the other
side of the back of mice. As shown in Figure 6, the signal of the
Figure 6. In vivo fluorescent imaging of mice injected with PBS/NE
(λex = 561 nm).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c08956.
■
sı
NE group increased in the red channel over time, but there was
no significant change in the control group. We believe that the
probe can be considered as an effective tool for detecting NE
in vivo as a good clinical application prospect in quantitative
analysis of neurotransmitters and signal pathways.
Experimental details and additional spectroscopic data
(PDF)
Norepinephrine Release Imaging with Fluoxetine.
Studies have shown fluoxetine as a selective serotonin reuptake
inhibitor can enhance serotonin neurotransmission to produce
antidepressant effects as fluoxetine,19 and it can also increase
dopamine and extracellular norepinephrine levels in the
hypothalamus,20 cortex,21 and prefrontal cortex.22 Currently,
fluoxetine is an effective clinical antidepressant on basal and
stress-induced norepinephrine (NE) release.23 Therefore, in
the following experiment, we carried out the norepinephrine
release imaging experiment with fluoxetine intervention. We
injected fluoxetine (10 mg/kg) intraperitoneally into the rats;
after 40 min of surgical exposure of the brain,24 the probe (200
μM) was directly added to the brain and then imaged.
Compared with the rats given the same volume of saline
intraperitoneally, the rats treated with fluoxetine had
significantly higher norepinephrine levels in the brain as the
control group (Figure 7). The results provided scientific data
for the screening and efficacy of antidepressant drugs in a clinic
in the future. At this point, we will study the intervention and
treatment of various antidepressants under all kinds of
depression through specific fluorescent probe bioimaging and
do in-depth research on the mechanism so as to provide a
powerful database for the development of effective anti-
depressants.
AUTHOR INFORMATION
Corresponding Author
■
Caixia Yin − Key Laboratory of Chemical Biology and Molecular
Engineering of Ministry of Education, Institute of Molecular
Science, Shanxi University, Taiyuan 030006, China;
orcid.org/0000-0001-5548-6333; Email: yincx@
sxu.edu.cn
Authors
Na Zhou − Key Laboratory of Chemical Biology and Molecular
Engineering of Ministry of Education, Institute of Molecular
Science, Shanxi University, Taiyuan 030006, China
Fangjun Huo − Research Institute of Applied Chemistry, Shanxi
University, Taiyuan 030006, China
Yongkang Yue − Key Laboratory of Chemical Biology and
Molecular Engineering of Ministry of Education, Institute of
Molecular Science, Shanxi University, Taiyuan 030006, China;
orcid.org/0000-0001-7958-0500
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c08956
Author Contributions
This manuscript was written through contributions from each
author. Each author approved the final version of this
manuscript.
CONCLUSION
■
Notes
In view of the challenge of developing specific NE probes, we
used this strategy: the fluorophore protected by the leaving
The authors declare no competing financial interest.
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(15) Yue, Y. K.; Huo, F. J.; Yin, C. X. Noradrenaline-specific,
efficient visualization in brain tissue triggered by unique cascade
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(16) Westerink, R. H.; Ewing, A. G. The PC12 cell as model for
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(17) Zhang, L.; Liu, X. A.; Gillis, K. D.; Glass, T. E. A high-affinity
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ACKNOWLEDGMENTS
■
We thank the National Natural Science Foundation of China
(Nos. 22074084, 21775096, 21907062), One Hundred People
Plan of Shanxi Province, Shanxi Province “1331 project” Key
Innovation Team Construction Plan Cultivation Team (2018-
CT-1), 2018 Xiangyuan County Solid Waste Comprehensive
Utilization Science and Technology Project (2018XYSDJS-
05), Shanxi Province Foundation for Returness (2017-026),
Shanxi Collaborative Innovation Center of High Value-Added
Utilization of Coal-Related Wastes (2015-10-B3), Shanxi
Province Foundation for Selected (No. 2019), Innovative
Talents of Higher Education Institutions of Shanxi, Scientific
and Technological Innovation Programs of Higher Education
Institutions in Shanxi (2019L0031), Key R&D Program of
Shanxi Province (201903D421069), Shanxi Province Science
Foundation (No. 201901D111015), and China Institute for
Radiation Production and Scientific Instrument Center of
Shanxi University (201512).
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