Lasers Med Sci
DOI 10.1007/s10103-013-1511-2
ORIGINAL ARTICLE
Evidence for A1 and A3 receptors mediating
adenosine-induced intracellular calcium release in the dorsal
root ganglion neurons by using confocal microscopy imaging
Liqin Zheng & Jiangxu Chen & Yimei Huang &
Yuhua Wang & Hongqin Yang & Yanding Zhang &
Shusen Xie
Received: 1 May 2013 / Accepted: 5 December 2013
# Springer-Verlag London 2013
Abstract Adenosine exerts a key role in analgesia. In the
present study, adenosine-induced Ca2+ responses were re-
vealed by using confocal microscopy imaging in the rat dorsal
root ganglia (DRG) neurons in vitro. Our results showed that
adenosine could evoke increases in the intracellular Ca2+
concentration in the DRG neurons. In addition, by application
of selective receptor antagonists, two types of receptors, A1R
and A3R, were identified to be involved in the adenosine-
induced Ca2+ release from intracellular stores in neurons.
Altogether, these results suggest that confocal microscopy
imaging combined with fluorescent dyes could help to detect
the analgesic-induced ion signaling in single cell.
Keywords Adenosine . Adenosine receptors . DRG neurons .
Confocal microscopy imaging
Liqin Zheng, Jiangxu Chen, and Yimei Huang contributed equally to this
work.
L. Zheng : J. Chen : Y. Huang : Y. Wang : H. Yang : S. Xie (*)
Institute of Laser and Optoelectronics Technology, Fujian Provincial
Key Laboratory for Photonics Technology, Key Laboratory of
OptoElectronic Science and Technology for Medicine of Ministry of
Education, Collage of Photonic and Electronic Engineering, Fujian
Normal University, Fuzhou 350007, China
e-mail: ssxie@fjnu.edu.cn
L. Zheng : Y. Zhang (*)
Fujian Key Laboratory of Developmental and Neural Biology,
College of Life Sciences, Fujian Normal University, Fuzhou 350108,
China
e-mail: ydzhang@fjnu.edu.cn
Introduction
Dorsal root ganglion (DRG) neurons are the primary sensory
neurons. Almost all ion channels as well as a wealth of
receptors such as opioid receptors [1], the GABAB receptors
[2], and sensory neuron-specific G-coupled receptors [3] are
assembled in the DRG neurons. And as for DRG neurons in
cultured sharing similarities with that in vivo [4–9], DRG
neurons cultured in vitro were used as a cellular model to
explore many physiological and pharmacological studies.
Adenosine is an endogenous neuromodulator and can
mainly be generated from AMP by 5′-nucleotidase.
Adenosine exerts its effects by activating four adenosine
receptors: adenosine A1 receptor (A1R), adenosine A2A recep-
tor (A2AR), adenosine A2B receptor (A2BR), and adenosine A3
receptor (A3R) [10–12]. With the development of agonists and
antagonists, adenosine and its receptors have been reported
that they were closely related to the transmission and modu-
lation of pain signals both in the central nervous system and
the peripheral nervous system [13–24]. And studies about
adenosine and its receptors on single cell such as neuron and
astrocyte have also been performed previously. Lao et al.
examined the effect of adenosine on glutamatergic miniature
excitatory postsynaptic currents in substantia gelatinosa neu-
rons, and found that adenosine could affect the glutamatergic
miniature excitatory postsynaptic currents in a dose-
dependent manner through A1R [25]. Doengi et al. have found
that A2AR contributed to the release of Ca2+ from the intra-
cellular stores such as mitochondria, endoplasmic reticulum to
the cytosol in astrocytes in the olfactory bulb slices [26]. A2BR
has also been reported to mediate the adenosine-induced Ca2+
increases in the astrocytes in hippocampal brain slices [27,
28]. However, some reports showed that adenosine could not

Fig. 1 Schematic of confocal microscopy imaging experimental setup
evoke Ca2+ increase in cultured astrocytes from the spinal
cord and hippocampus [29, 30]. Therefore, we propose that
the mechanisms of adenosine and its receptors to modulate the
Ca2+ responses are diverse for different kinds of cells. In this
study, we used confocal microscopy to record the Ca2+ re-
sponses in cultured DRG neurons from adult rats after being
treated with adenosine and adenosine receptor antagonists. We
compared the differences between control group (neurons
being treated with Neuralbasal-A media) and experimental
group (neurons being treated with adenosine or adenosine
receptor antagonists) to determine whether the method can
be used to record the Ca2+ responses and to explore whether
adenosine can evoke Ca2+ responses in DRG neurons and
which adenosine receptor mediates the adenosine signaling
transmission.
Fig. 2 Dorsal root ganglion neurons cultured in vitro for 3 days and identified with β3-tubulin antibody. The cells with green fluorescence were neurons.
The right figure was the enlargement of the rectangle in the left figure
Lasers Med Sci
Materials and methods
Cell culture and antibody staining
Primary DRG neurons were isolated from rats (Sprague
Dawley; 8–12 weeks of age) and plated on the 35-mm cell
culture dishes (801002, Nest) with poly-D-lysine (P7280,
Sigma) and laminin (L2020, Sigma) mainly according to the
previous report [31]. Neurons were cultured in Neuralbasal-A
media (10888-022, Invitrogen) supplemented with 2 % B27
(17504-044, Invitrogen), 100 units/ml Pen-strep-neo (15140-
122, Invitrogen) and 2-mM GlutaMax™ (35050-061,
Invitrogen), and were grown at 37 °C in 5 % CO2 incubator.
DRG neurons cultured for 3 days in vitro (DIV) were used
for antibody staining according to the protocols. Briefly, neu-
rons were kept at 4 °C for 1 h in 4 % paraformaldehyde in
phosphate buffered saline (PBS, pH 7.4) and were loaded with
0.3 % Triton-X-100 for 10 min after rinsing with PBS three
times. Then, neurons were incubated in blocking solution
(1 % normal goat serum) for 30 min at room temperature.
Afterwards, neurons were incubated with the primary anti-
body (mouse anti-β3 tubulin polyclonal antibody,1:200, sc-
80005, Santa Cruz) overnight at 4 °C after removing the
blocking solution. And then neurons were incubated with
the secondary antibody (goat-anti-mouse-fluorescein isothio-
cyanate (FITC) IgG, sc-2010, Santa Cruz) for 2 h after rinsing
with PBS three times. At last, neurons were mounted with
Ultra Cruz TM Mounting Medium (sc-24941, Santa Cruz).
Ca2+ imaging
DRG neurons cultured for 3 DIV were loaded with 5-μM
Fluo-3/AM (F-14218, Invitrogen) and 0.02 % Pluronic F-127
Lasers Med Sci
Fig. 3 Ca2+ dynamic changes after application of adenosine in cultured
DRG neurons. a confocal Ca2+ imaging, scale bar=10 μm. b Curves of
Ca2+ responses. Ca2+ transients evoked by adenosine occurred at different
time in DRG neurons. In this figure and the following figure, F/Fmax
indicates fluorescence normalization. c Mean ΔF of adenosine-induced
Ca2+ transients in DRG neurons. In this figure and the following figure,
bars are mean ± SD, n indicates the number of cells investigated. Ca2+
changes induced by adenosine were significantly major than control
group. * indicates P<0.05
Lasers Med Sci
 Lasers Med Sci
ƒ Fig. 4 Effect of adenosine receptor antagonists. a Curves of Ca2+ re-
sponses evoked by adenosine receptor antagonists. DPCPX and VUF-
5574 blocked Ca2+ transients evoked by adenosine in neurons, while
ZM241385 and Alloxazine failed to block Ca2+ transients. b Statistical
analyses of ΔF, * indicates P<0.05, ** indicates P>0.05
(P6866, Invitrogen) at 37 °C for 30 min. Then, neurons were
washed with PBS three times, added with Tyrode solution
(150-mM NaCl, 5-mM KCl, 10-μM EGTA, 1-mM
MgSO4•7H2O, 10-mM D-glucose, 10-mM HEPES, pH 7.4)
and incubated at room temperature for 30 min. When imaging
using confocal microscopy, except for the control group,
adenosine (500 μM; A9251, Sigma) or four different adeno-
sine receptor antagonists solution (8-cyclopentyl-1,3-
dipropyl-xanthine (DPCPX), ZM241385, alloxazine, and
VUF-5574 are receptor antagonists corresponding to A1R,
A2AR, A2BR, and A3R, respectively; all were purchased from
Sigma) were added into the center of the culture dishes re-
spectively. Cells of the control group were loaded with the
same amount of Neuralbasal-A media. Adenosine and aden-
osine receptor antagonists were all dissolved in Neuralbasal-A
media.
Confocal microscopy imaging
The confocal microscopy used in this study was an Axiovert
200 microscopy (Zeiss LSM 510 META), which has been
described previously [32, 33]. In brief, as shown in Fig. 1,
both the two fluorescent dyes FITC and Fluo-3 used in the
study were excited with light at 488 nm from an argon laser. In
order to focus the excitation beam and collect the backward
signals, a plan-Neofluar 40×(N.A.=0.75) dry objective (Zeiss)
was employed. A main dichroic beam splitter (MDBS) was
employed in directing the backward signals to the META
detector. The META detector with 32-gated photon counting
modulate was used to collect x–y images at different emission
wavelengths. The emission wavelength of 520–530 nm was
used to detect FITC, and emission wavelength of 510–560 nm
was used to detect Fluo-3. In front of the META detector, there
was a filter (Zeiss HFT 488) which was used to ensure that
excitation light was filtered out and only emission signals were
recorded. All images have a 12-bit pixel depth.
Data and statistics
Within a time series, the fluorescence signals of intracellular
Ca2+ were assessed in the regions of interest (ROI) selecting in
the soma of DRG neurons stained with Fluo-3. The Ca2+
responses curves of ROI were presented as F/Fmax. Ca2+
changes after application adenosine or adenosine receptor
antagonists were given as relative fluorescence changes
(ΔF). F and Fmax indicate the fluorescence intensity of Ca2+
signaling at each time recorded and Ca 2+ transient,
respectively, and Fresting means the resting Ca2+ fluorescence
intensity. The major of ΔF, the more significant changes in the
concentration of intracellular calcium ([Ca2+]i) after applica-
tion adenosine or adenosine receptor antagonists. On the other
hand, the minor of ΔF, the less changes in the [Ca2+]i.

.
ΔF ¼ F max − F resting F max
All experimental measurements were presented as means ±
SD, with n indicating the number of individual DRG neurons.
Statistical comparisons were made using one-way ANOVA,
Students-Neuman-Keuls multiple comparisons (SPSS,
Version 16.0, http://www.spss.com). P<0.05 was considered
to be significant.
Results and discussion
According to previous studies, DRG neurons are usually
divided into three types based on the neuronal size: small,
medium, and large. As shown in Fig. 2, almost all cultured
neurons we obtained were presented with about 20-μm diam-
eter soma and well developed neuritic trees. Passmore GM
have reported that DRG neurons cultured in vitro might be a
model system for identifying novel analgesic targets as they
could share characteristics with nociceptors especially the
neurons with a soma diameter less than 30 μm [34]. So it
indicated that the neurons we cultured could be used to detect
novel analgesics for the treatment of pain.
Adenosine is considered to be a therapeutic agent for pain
as its antinociceptive effects [35, 36]. In this work, the spatial
and temporal changes of adenosine-induced intracellular Ca2+
responses in cultured rat DRG neurons were detected using
time-series confocal Ca2+ imaging. As shown in Fig. 3a, the
confocal Ca2+ images of the resting and activated condition
after loading with adenosine were captured. The Ca2+ fluores-
cence intensity of the neurons treated with adenosine was
significantly changed when compared with the resting fluo-
rescence, while Neuralbasal-A media had no effect on the
Ca2+ fluorescence of the control group. And temporal changes
of intracellular Ca2+ responses after application of adenosine
were also obtained. In Fig. 3b, the F/Fmax of four ROI were
shown. From the figure, we could see that adenosine could
evoke Ca2+ transients at different time. The statistics of the
effect of adenosine was quantified, as shown in Fig. 3c. The
results confirmed that the neurons loaded with adenosine had
higher ΔF (ΔFadenosine=0.2808±0.0185, ΔFcontrol=0.0419±
0.0084, P<0.05). In this study, extracellular Ca2+ could be
chelated by EGTA contained in the Tyrode solution, so the
major intracellular Ca2+ signals that we got might be due to the
calcium release from the intracellular Ca2+ stores such as the
mitochondria and endoplastic reticulum. Thayer et al. [37]

have showed that a larger Ca2+ signals might be the result of a
collapse of mitochondrial membrane potential, and, as we
know, that Δψm is an important parameter of cell apoptosis.
So, we propose that high-dose adenosine result in the apopto-
sis of neurons which contributes to the fall of Δψm, and thus
result in evoking a Ca2+ rise in the DRG neurons because of
the releasing of Ca 2+ from the mitochondria into the
cytoplasm.
Based on confocal microscopy imaging, the effects of four
different selective adenosine receptor antagonists on Ca2+
responses in DRG neurons was further performed to detect
which adenosine receptor was involved in the adenosine sig-
naling transmission. When imaging, adenosine receptor
blockers were added into the neurons prior to 500-μM aden-
osine in order to avoid evoking Ca2+ transients by adenosine
in a short time. As shown in Fig. 4a, the F/Fmax curves of ROI
after application of adenosine receptor blockers were shown.
From the figure, we could see that the Ca2+ transients evoked
by adenosine were significantly blocked by the presence of
A1R antagonist DPCPX and A3R antagonist VUF-5574 in
most of the neurons investigated, while A2AR antagonist
ZM241385 and A2BR antagonist Alloxazine failed to block
Ca2+ transients evoked by adenosine. The ΔF was further
calculated to confirm the results, as shown in Fig. 4b. The
ΔF of DPCPX and VUF-5574 was 0.0868±0.0323 and 0.057
±0.0131, respectively, both of which were significantly lower
than the other groups (ΔFZM241385 = 0.5248 ± 0.1381,
ΔFAlloxazine=0.6092±0.0979) (P<0.05). It demonstrated that
both A1R and A3R might participate in the adenosine signal
transmission in DRG neurons. Many reports showed that A1R
participated in the pain signal transmission [13–16], but few
were reported about A3R [23]. Our results provided strong
evidence that not only A1R but also A3R might be involved in
the pain signal transmission.
Conclusion
In summary, adenosine could act as a neurotransmitter to
evoke the intracellular Ca2+ signaling in DRG neurons mainly
through A1R and A3R. And confocal microscopy imaging
combined with fluorescent dyes has been demonstrated to be
an effective technique to monitor the adenosine-induced Ca2+
dynamics in single cell. We foresee potential application of the
method as aid to detect the analgesics-induced ion signaling in
single cell in the future.
Acknowledgments This work was supported by the Program for
Changjiang Scholars and Innovative Research Team in University (Grant
No. IRT 1115), the National Natural Science Foundation of China (Grant
No. 61335011 and Grant No. 60978071), the Natural Science Foundation
of Fujian Province (Grant No. 2010 J01322) and the Fujian Province
Educational Project B (Grant No. JB11023).
Lasers Med Sci
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