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PNAS 2025 Vol. 122 No. 3 e2406741122 https://doi.org/10.1073/pnas.2406741122 1 of 11
RESEARCH ARTICLE
|
Significance
The neurotransmission between
dorsal root ganglion (DRG) and
spinal cord neurons is essential for
pain sensation. Mounting evidence
has established the crucial roles of
action potential (AP)-evoked
neurotransmission from DRG
neurons in chronic pain. However,
the function and mechanism of
AP-independent
neurotransmission are not fully
understood. Here, we observed
frequent spontaneous
microdomain Ca2+ (smCa) activities
across DRG neurons’ somata and
axons independent of APs. These
smCa activities, mediated by
spontaneous activation of TRPA1,
triggered continuous
neurotransmission from DRG to
spinal cord, which was
substantially elevated in
inammatory pain conditions.
Importantly, inhibiting smCa or the
associated continuous
neurotransmission alleviated
hyperalgesia. Our study may have
identied a unique mechanism
underlying nociceptive
sensitization in chronic pain.
The authors declare no competing interest.
This article is a PNAS Direct Submission.
Copyright © 2025 the Author(s). Published by PNAS.
This article is distributed under Creative Commons
Attribution- NonCommercial- NoDerivatives License 4.0
(CC BY- NC- ND).
1To whom correspondence may be addressed. Email:
zhangzhuoyu7@163.com, changhewang@xjtu.edu.cn,
zuyingchai@gmail.com, or huangrong@xjtu.edu.cn.
2Z.Z., J.Y., and J.H. contributed equally to this work.
This article contains supporting information online at
https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.
2406741122/- /DCSupplemental.
Published January 17, 2025.
CELL BIOLOGY
Action potential–independent spontaneous microdomain
Ca2+ transients–mediated continuous neurotransmission
regulates hyperalgesia
ZhuoyuZhanga,b,1,2 , JingyuYaoa,2 , JingxiaoHuoa,2, RuolinWanga, XuetingDuana, YangChena, HuadongXua, ChangheWanga,c,1 , ZuyingChaid,1 ,
and RongHuanga,1
Aliations are included on p. 10.
Edited by Hee- Sup Shin, Institute for Basic Science, Daejeon, Korea (South); received April 3, 2024; accepted December 9, 2024
Neurotransmitters and neuromodulators can be released via either action poten-
tial (AP)–evoked transient or AP- independent continuous neurotransmission. e
elevated AP- evoked neurotransmission in the primary sensory neurons plays cru-
cial roles in hyperalgesia. However, whether and how the AP- independent con-
tinuous neurotransmission contributes to hyperalgesia remains largely unknown.
Here, we show that primary sensory dorsal root ganglion (DRG) neurons exhibit
frequent spontaneous microdomain Ca2+ (smCa) activities independent of APs
across the cell bodies and axons, which are mediated by the spontaneous opening of
TRPA1 channels and trigger continuous neurotransmission via the cyclic adenosine
monophosphate- protein kinase A signaling pathway. More importantly, the frequency
of smCa activity and its triggered continuous neurotransmission in DRG neurons
increased dramatically in mice experiencing inflammatory pain, inhibition of which
alleviates hyperalgesia. Collectively, this work revealed the AP- independent contin-
uous neurotransmission triggered by smCa activities in DRG neurons, which may
serve as a unique mechanism underlying the nociceptive sensitization in hyperalgesia
and offer a potential target for the treatment of chronic pain.
continuous neurotransmission | hyperalgesia | sensory dorsal root ganglion neurons |
spontaneous microdomain Ca2+ activities | TRPA1
Chronic pain is a multifaceted and persistent disease that aects more than 30% of people
all over the world and costs billions of dollars every year ( 1 , 2 ). Pain signals start from
primary sensory dorsal root ganglion (DRG) neurons ( 3 – 5 ), which function as environ-
mental detectors (heat, cold, mechanical force, etc.) and transmit sensory information
from the periphery to the central nervous system. e communication between DRG
neurons and central spinal cord neurons through neurotransmission is essential for pain
processing. In addition to pain sensation in physiological conditions, DRG neurons also
play a key role in the progression and maintenance of chronic pain ( 6 , 7 ). However, the
molecular and cellular mechanisms remain not fully understood.
A large population of neurotransmitters (glutamate, ATP, etc.) and neuromodulators
[Substance P, CGRP, neuropeptide Y (NPY), etc.] are secreted from DRG neurons and
involved in the development and maintenance of chronic pain ( 8 , 9 ). e concentra-
tions of these neurotransmitters and neuromodulators in DRG and spinal cord are
greatly elevated in individuals experiencing chronic pain ( 10 – 15 ). Neurotransmitters
and neuromodulators can be released via either action potential (AP)–evoked or
AP-independent neurotransmission. Mounting evidence has established the crucial
roles of AP-coupled neurotransmission from DRG neurons in chronic pain. However,
whether and how AP-independent continuous neurotransmission contributes to
chronic pain remains largely unknown.
In the current study, we observed frequent spontaneous microdomain Ca2+ (smCa)
activities independent of APs across the cell bodies and axons of primary sensory DRG
neurons using high-resolution real-time total internal reection uorescence (TIRF) imag-
ing. e smCa activities trigger continuous neurotransmission in a protein kinase A
(PKA)-dependent manner. Importantly, the smCa-induced continuous neurotransmission
in DRG neurons increased dramatically in chronic pain, inhibition of which alleviates
hyperalgesia. Collectively, this study denes the AP-independent continuous neurotrans-
mission triggered by smCa activities as a possible unique mechanism underlying the
nociceptive sensitization of primary sensory neurons, providing a potential therapeutic
target for the treatment of chronic pain.
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Fig. 1. smCa activities in DRG neurons. (A, Upper) A TIRF image of cultured DRG neuron transfected with Lck- GCaMP5g; (Lower) A cartoon illustrating that the
Lck- GCaMP5g (black) binds with Ca2+ and emits green uorescence. (Scale bar, 10 μm.) (B and C) The surface plot of the Ca2+ signal from a typical ROI (dened
in Materials and Methods) in A at inactive and active states. (D) The uorescence intensity of an active ROI in 1 min with the hot image shown below. The smCa
events (dened in methods) are indicated by black bars at the Top. (E) The whole- cell surface plot of the Ca2+ signal from the DRG neuron in A. (F) The hot image
of the Ca2+ signals before and after the treatment of EGTA- AM (10 μM) from 12 active ROIs in the DRG neuron shown in A. (G) Quantication for the total active
ROIs per cell per minute before and after the treatment of EGTA- AM. (H) The smCa events from 11 cells before and after the treatment of EGTA- AM. Each row
shows the total smCa events in 1 min from one DRG neuron. (I) Quantication for the total smCa events per cell per minute before and after the treatment of
EGTA- AM. (J–M) The same as F–I, but replace EGTA- AM with BAPTA- AM (10 μM). Data were shown as a violin plot with all data points included. Paired student’s
t test. ns, not signicant; *P < 0.05; **P < 0.01.
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PNAS 2025 Vol. 122 No. 3 e2406741122 https://doi.org/10.1073/pnas.2406741122 3 of 11
Results
smCa Activities in Sensory DRG Neurons. e real- time TIRF
imaging (16, 17) of a membrane- localized Ca2+- indicator, Lck-
GCaMP5g (18), was used to simultaneously monitor local Ca2+
signals beneath the plasma membrane in both the soma and
axons of individual primary sensory DRG neurons. We strikingly
observed frequent spontaneous Ca2+ activities across the somatic
and axonal regions of DRG neurons in normal Ca2+- containing
bath solution (Fig.1 A–E, SI Appendix, Fig.S1 A and B, and
Movies S1–S4). ese spontaneous Ca
2+
activities were conned to
a restrained area without spreading out. In addition, the number of
active regions of interest (ROIs) and ring rate of the spontaneous
Ca
2+
signals in each DRG neuron were signicantly decreased after
incubation with N,N’- [1,2- ethanediylbis(oxy- 2,1- phenylene)]
bis[N- [2- [(acetyloxy)methoxy]- 2- oxoethyl]- 1,1’- bis[(acetyloxy)
IK
H
G
F
CDE
B
A
J
Fig. 2. SmCa is dependent on extra-
cellular Ca
2+
and TRPA1 channels.
(A, Left) A TIRF image of cultured
DRG neuron transfected with Lck-
GCaMP5g; (Right) The whole- cell
surface plot of the Ca
2+
signal from
the DRG neuron in the Left panel
of A. (Scale bar, 10 μm.) (B) The hot
image of the Ca
2+
signals from 12
active smCa ROIs in the DRG neu-
ron shown in A, in 2 mM, 0 mM,
and 2 mM Ca2+- containing baths,
respectively. (C) Quantication for
the total active ROIs per cell per
minute in 2 mM, 0 mM, and 2 mM
Ca2+- containing baths, respectively.
(D) Quantication for the total active
ROIs per cell per minute before and
after whole- cell voltage clamping to
−70 mV. (E, Left) Quantication for
the total active ROIs per cell per
minute before and after the treat-
ment of cocktail of inhibitors [1 μM
TTX (antagonist of voltage- gated
sodium channel), 1 μM GVIA (antag-
onist of voltage- gated N- type Ca2+
channel), 5 μM Nifedipine (antag-
onist of voltage- gated L- type Ca2+
channel), and 200 μM CdCl2 (the
pore blocker of all VGCCs)]. (Right)
Quantication for the total active
ROIs per cell per min before and
after the treatment of 1 μM TTA- A2
(antagonist of voltage- gated T- type
Ca2+ channel). (F) Quantication for
the total active ROIs per cell per
minute before and after the treat-
ment of broad- spectrum antagonist
of TRP channels (50 μM RR, Rutheni-
um Red). (G) Quantication for the
total active ROIs per cell per minute
before and after the treatment of
TRPA1- specic antagonist, 1 μM
A967079 (Left) or 20 μM HC030031
(Right), respectively. (H) Quantica-
tion for the total active ROIs per
cell per minute before and after
the treatment of TRPV1- specic
antagonist, 10 μM AMG9810. (I)
Quantication showing the total
active ROIs per cell per minute (Left)
and total smCa events per cell per
minute (Right) in WT, TRPA1−/− and
TRPV1
−/−
DRG neurons, respectively.
(J) Quantication showing the total
active ROIs per cell per minute (Left)
and total smCa events per cell per
minute (Right) in control and TRPA1
overexpression DRG neurons, re-
spectively. (K) A cartoon illustrating
that smCa activity is mediated by
the Ca2+ inux through TRPA1 chan-
nel. Data were shown as a violin plot
with all data points included. Paired
student’s t test for D–H. Student’s t
test for J. One- way ANOVA with
a post hoc test enabling multiple
comparisons for C and I. ns, not
signicant; **P < 0.01; ***P < 0.001.
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B
CDE
G
J
I
H
F
A
Fig. 3. SmCa regulates spontaneous quantal vesicle release in the somata of DRG neurons. (A, Upper) the TIRF image of a 24 h cultured DRG neuron transfected
with NPY- pHluorin; (Lower) A cartoon illustrating that NPY- pHluorin emits green uorescence when vesicle lumen’s pH increases from 5.5 to 7.4 during fusion.
(Scale bar, 10 μm.) (B) The uorescence intensity versus time and the TIRF image montage showing a single quantal vesicle release process from three events
marked in A, which are aligned by the onset point of each release. Each release event happens spontaneously and randomly at dierent time in 1 min recording. (C)
Quantication for the total spontaneous quantal release events per cell per minute in 2 mM Ca
2+
and 0 mM Ca
2+
- containing baths, respectively. (D) Quantication
for the total spontaneous quantal release events per cell per minute, before and after the treatment of RR (Left) or A967079 (Right). (E) Quantication for the total
spontaneous quantal release events per cell per minute in WT and TRPA1−/− DRG neurons. (F, Upper) the TIRF image of a 24 h cultured DRG neuron transfected
with Spy- pHluorin; (Lower) A cartoon illustrating that Spy- pHluorin emits green uorescence when vesicle lumen’s pH increases from 5.5 to 7.4 during fusion.
(Scale bar, 10 μm.) (G) The uorescence intensity versus time and the TIRF image montage showing a single quantal vesicle release process from three events
marked in F. (H) Quantication for the total spontaneous quantal release events per cell per minute in 2 mM Ca2+ and 0 mM Ca2+- containing baths, respectively.
(I) Quantication for the total spontaneous quantal release events per cell per minute, before and after the treatment of RR (Left) or A967079 (Right). (J) A cartoon
illustrating that spontaneous Ca2+ inux through the TRPA1 channel (smCa) regulates spontaneous quantal vesicle release. Data were shown as a violin plot
with all points included. Paired student’s t test for C/D/H/I. Student’s t test for E. *P < 0.05; **P < 0.01; ***P < 0.001.
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methyl] ester- glycine (BAPTA- AM), a fast and potent Ca2+-
chelator (3, 19), but not aected by the slow Ca2+- chelator
3,12- bis[2- [(acetyloxy)methoxy]- 2- oxoethyl]- 6,9- dioxa- 3,12-
diazatetradecanedioic acid, 1,14- bis[(acetyloxy)methyl] ester
(EGTA- AM) (Fig.1 F–M), indicating that the spontaneous Ca2+
activities in DRG neurons are microdomain Ca2+ (smCa) signals.
To determine the origin of the smCa signals, we removed the
extracellular Ca2+ or depleted the intracellular Ca2+ store with
2- Aminoethyl diphenylborinate (2- APB). e smCa signals in the
somata and axons of DRG neurons disappeared in Ca2+- free bath
solution and then fully recovered after switching back to normal
Ca2+- containing solution (Fig.2 A–C and SIAppendix, Fig.S1
C–E), suggesting that the smCa signals originate from Ca
2+
- inux
via channels on the plasma membrane of DRG neurons, instead
of the intracellular Ca2+ store because 2- APB did not aect smCa
signals (SIAppendix, Fig.S2 A and B).
A
C
E
G
IJ
H
F
D
B
Fig. 4. SmCa regulates continuous miniature synaptic transmission from DRG to spinal dorsal horn neurons. (A) A light- eld image showing patch- clamp
recording on postsynaptic spinal dorsal horn (DH) neurons cocultured with DRG neurons. (Scale bar, 10 μm.) (B) A cartoon illustrates the recording for mEPSC.
(C) The typical mEPSC traces from cocultured DRG- dorsal horn synapses in 2 mM Ca2+ and 0 mM Ca2+- containing baths, respectively. (D) Quantication for the
normalized mEPSC event number per minute (Left) and the amplitude (pA, Right) in 2 mM Ca2+ and 0 mM Ca2+- containing baths shown in C. (E) The typical mEPSC
traces from cocultured DRG- dorsal horn synapses before and after RR treatment. (F) Quantication for the normalized mEPSC event number per minute (Left)
and the amplitude (pA, Right), before and after RR treatment shown in E. (G) The typical mEPSC traces from cocultured DRG- dorsal horn synapses before and
after A967079 treatment. (H) Quantication for the normalized mEPSC event number per minute (Left) and the amplitude (pA, Right), before and after A967079
treatment shown in G. (I) The typical mEPSC traces from WT spinal dorsal horn neurons cocultured with WT and TRPA1−/− DRG neurons. (J) Quantication for the
mEPSC event number per minute (Left) and the amplitude (pA, Right) shown in I. Data were shown as a violin plot with all points included. Paired student’s t test
for D/F/H. Student’s t test for J. ns, not signicant; *P < 0.05; **P < 0.01; ***P < 0.001.
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A
B
C
ED
FHJ
GIK
Fig. 5. SmCa regulates spontaneous vesicle release via PKA. (A) A TIRF image of a 24 h cultured DRG neuron transfected with NPY- pHluorin. (Scale bar, 10 μm.)
(B) A cartoon illustrating that NPY- pHluorin emits green uorescence when the vesicle lumen’s pH increases from 5.5 to 7.4 during fusion. (C) Quantication
for total spontaneous quantal release events per cell per minute by TIRF imaging, before and after treatment of the PKA antagonist (2 μM H89). (D) The typical
mEPSC traces from cocultured DRG and dorsal horn neurons before and after H89 treatment. (E) Quantication for the normalized mEPSC event number per
minute (Left) and the amplitude (pA, Right) before and after H89 treatment. (F) Typical mEPSC traces recorded from cocultured DRG and dorsal horn neurons,
rst in normal Ca
2+
- containing bath solution (Top), then TRPA1 antagonist (A967079) was applied (Middle), and last PKA inhibitor (H89) was added on top of TRPA1
antagonist (Bottom). (G) Quantication for the normalized mEPSC event number per minute (Left) and the amplitude (pA, Right) in F. (H) Typical mEPSC traces
recorded from cocultured DRG and dorsal horn neurons, rst in normal Ca2+- containing bath solution (Top), then PKA inhibitor (H89) was applied (Middle), and
last TRPA1 antagonist (A967079) was added on top of PKA inhibitor (Bottom). (I) Quantication for the normalized mEPSC event number per minute (Left) and the
amplitude (pA, Right) in H. (J) Typical mEPSC traces recorded from cocultured DRG and dorsal horn neurons, rst in normal Ca2+- containing bath solution (Top),
then TRPA1 antagonist (A967079) was applied (Middle), and last PKA agonist (25 μM sp- cAMP) was added on top of TRPA1 antagonist (Bottom). (K) Quantication
for the normalized mEPSC event number per minute (Left) and the amplitude (pA, Right) in J. Data were shown as a violin plot with all points included. Paired
student’s t test for C and E. One- way ANOVA with a post hoc test enabling multiple comparisons for G, I, and K. ns, not signicant. *P < 0.05; **P < 0.01; ***P < 0.001.
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TRPA1 Mediates smCa Activities in DRG Neurons. To determine
the Ca2+ channel responsible for smCa signals, we rst clamped
the membrane potential of DRG neurons at −70 mV to block
the activity of voltage- gated Ca2+ channels (VGCCs) and found
that it did not aect the smCa signals (Fig.2D and SIAppendix,
Fig.S2 C and D). In addition, the cocktail of inhibitors (including
1 μM TTX for voltage- gated Na+ channel, 1 μM ω- conotoxin
GVIA for voltage- gated N- type Ca2+ channel, 5 μM nifedipine
for voltage- gated L- type Ca2+ channel and 200 μM CdCl2 for
all VGCCs) and 1 μM TTA- A2 (antagonist of voltage- gated
T- type Ca2+ channel) (3, 19) showed no eect on the smCa
as well (Fig.2E and SI Appendix, Fig.S2 E–H), excluding the
contribution of VGCCs and supporting the AP- independency of
these smCa signals. Next, we tried ruthenium red (RR, 50 μM),
a nonspecic broad- spectrum antagonist for all transient receptor
potential (TRP) channels. Surprisingly, RR almost eliminated the
smCa signals (Fig.2F and SIAppendix, Fig.S2 I and J), suggesting
the involvement of TRP channels in smCa activities. Furthermore,
two specic inhibitors for TRPA1 (A967079, 1 μM or HC030031,
20 μM) blocked the smCa signals signicantly (Fig. 2G and
SIAppendix, Fig.S3), but the antagonist of TRPV1 (AMG9810,
10 μM) showed no eect (Fig.2H), suggesting an essential role
of TRPA1 in smCa signals. In addition, the spontaneous inward
membrane currents of DRG neurons at resting membrane
potential were also inhibited by TRPA1 antagonist (SIAppendix,
Fig.S1 F and G). Consistently, the number of active regions and
GI
D
B
A
C
E
HI J
F
Fig. 6. Spontaneous smCa- mediated continuous neurotransmission regulates chronic pain behavior. (A) A photograph showing the hind paw’s thickness
between the ipsilateral and contralateral sides after saline or CFA injection, respectively. (B) Quantication of the hind paw’s thickness between the ipsilateral
and contralateral side after saline or CFA injection, respectively. (C) The whole- cell surface plot of the spontaneous Ca2+ signals in DRG neurons from WT C57
injected with saline or CFA. (D) Quantication for the total smCa ROIs number per cell per minute in DRG neurons from WT C57 or TRPA1−/− mice injected with
saline and CFA, respectively. (E) The TIRF images of spontaneous vesicle release in DRG neurons from WT C57 mice injected with saline or CFA group. (Scale bar,
10 μm.) (F) Quantication for the spontaneous quantal vesicle release events per cell per minute in DRG neurons from WT C57 or TRPA1−/− mice injected with
saline and CFA, respectively. (G) Statistics of the latency of paw licking in hot plate behavior test for WT or TRPA1−/− mice injected with saline or CFA. (H) Statistics
of the latency of paw licking in hot plate behavior test for CFA- inamed WT mice 1- h after the acute hind paw injection of saline, 20 μM H89, or 200 μM H89.
(I) Statistics of the mechanical pain threshold in Von Frey behavior test for WT or TRPA1−/− mice injected with saline or CFA. (J) Statistics of the mechanical pain
threshold in Von Frey behavior test for CFA- inamed WT mice 1- h after the acute hind paw injection of saline, 20 μM H89, or 200 μM H89. Data were shown as
a violin plot with all points included. One- way ANOVA- test for H and J. Two- way ANOVA- test for G and I. Paired student’s t test for B. Student’s t test for D and F.
ns, not signicant; *P < 0.05; **P < 0.01; ***P < 0.001.
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ring rate of smCa signals in TRPA1−/−, but not TRPV1−/−, DRG
neurons were reduced signicantly compared with that of wild-
type (WT) DRG neurons (Fig.2I and SI Appendix, Fig.S4).
Contrarily, overexpression of TRPA1 in DRG neurons increased
the number of active regions and ring rate of smCa signals
dramatically (Fig.2J and SIAppendix, Fig.S5). Taken together,
these ndings demonstrate that the smCa signals in DRG neurons
are mediated by the spontaneous activation of TRPA1 channels
and independent of APs (Fig.2K).
SmCa Triggers Continuous Neurotransmission in Both the Somata
and Terminals of DRG Neurons.
As an important second messenger,
Ca2+ has diverse functions in dierent cellular procedures, especially
its crucial roles in neurotransmission (20, 21). To investigate the
function of smCa activities in the neurotransmission of DRG
neurons, we labeled the large dense- core vesicles and small vesicles
of DRG neurons with NPY- pHluorin and synaptophysin (Spy)-
pHluorin, respectively (3, 18, 22) (Fig.3 A and F). Consistent with
the smCa signals, we observed abundant spontaneous release events
of both NPY- and Spy- pHluorin labeled vesicles from DRG neurons
in Ca2+- containing bath solution by using high- resolution TIRF live-
imaging, indicated by abrupt uorescence increase followed by the
decrease to baseline (Fig.3 B and G and Movies S5 and S6), which
were diminished when switching to Ca2+- free bath solution (Fig.3
C and H). In addition, the spontaneous release of DRG neurons was
largely inhibited by BAPTA- AM, instead of EGTA- AM (SIAppendix,
Fig.S6 A–D). In contrast, clamping the membrane potential at −70
mV did not aect the spontaneous release, indicating that smCa,
but not spontaneous AP, is the trigger for the spontaneous vesicle
release in DRG neurons (SIAppendix, Fig.S6E). Furthermore,
the nonspecic TRP channel blocker (RR) and specic antagonist
of TRPA1 (A967079), shown to block smCa in DRG neurons
(Fig.2 F and G), largely inhibited the spontaneous release of
both NPY- and Spy- pHluorin labeled vesicles in DRG neurons
(Fig.3 D and I). Moreover, the spontaneous release events were
signicantly fewer in TRPA1−/− DRG neurons compared with
that in WT neurons (Fig.3E), further conrming the essential
role of TRPA1- mediated smCa in the spontaneous vesicle release
of DRG neurons (Fig.3J).
Since smCa activities exist not only in the somata but also in
the axons of DRG neurons (SI Appendix, Fig. S1 A–E ), we set out
to study the role of smCa activities in synaptic transmission of
DRG neurons cocultured with spinal dorsal horn neurons ( Fig. 4
A and B ). Consistent with our TIRF imaging data, the frequency
of miniature excitatory postsynaptic current (mEPSC) recorded
from the dorsal horn neurons was reduced remarkably after switch-
ing from the normal Ca
2+
-containing solution to a Ca
2+
-free solu-
tion ( Fig. 4 C and D ). Besides, the mEPSC events were also largely
inhibited by the nonspecic TRP channel blocker (RR) and the
specic TRPA1 channel antagonist (A967079) ( Fig. 4 E –H ).
Furthermore, the frequency of mEPSC events from WT dorsal
horn neurons cocultured with TRPA1−/− DRG neurons was sig-
nicantly lower than that cocultured with WT DRG neurons
( Fig. 4 I and J ). In addition, we recorded the mEPSCs of dorsal
horn neurons from adult fresh spinal cord slices with intact DRG
central terminals and found that the mEPSC events were also
inhibited by TRPA1 antagonist (SI Appendix, Fig. S6 F−H ). Taken
together, smCa triggers continuous neurotransmission in both the
somata and terminals of DRG neurons.
PKA Plays an Essential Role in smCa- Triggered Continuous
Neurotransmission. PKA and protein kinase C (PKC) are important
kinases known to be activated by Ca
2+
and function in spontaneous
synaptic transmission (23, 24). To identify the key player mediating
smCa- triggered continuous neurotransmission in DRG neurons,
we tested the specic inhibitors of PKA (H89, 2 μM) and PKC
(Bis, 1 μM) and found that blocking PKA showed no eect on the
smCa activities (SIAppendix, Fig.S7 A and B), while blocking PKC
reduced smCa activities in DRG neurons (SIAppendix, Fig.S7 C
and D), implying that PKA may be downstream of smCa activities.
Intriguingly, the spontaneous release of NPY- pHluorin labeled
vesicles was reduced signicantly by H89, the PKA antagonist
(Fig.5 A–C), suggesting the functional involvement of PKA in
smCa- triggered spontaneous neurotransmission in DRG neurons.
Consistently, H89 also blocked the mEPSC events recorded from
dorsal horn neurons cocultured with DRG neurons (Fig. 5 D
and E). To further conrm that PKA is the downstream protein
kinase of smCa activities, we rst inhibited the smCa signals
by using the TRPA1 antagonist (A967079) and then applied
H89 to inhibit PKA activity. e frequency of mEPSC events
recorded from spinal dorsal horn neurons cocultured with DRG
was reduced by A967079 as shown before (Fig.4 G and H), but
did not further decrease with the addition of H89 (Fig.5 F and
G). Similarly, A967079 did not further lower the mEPSC event
frequency after H89 application (Fig.5 H and I), suggesting that
smCa and PKA are in the same signaling pathway. Furthermore,
after inhibiting the smCa- triggered mEPSC with A967079, the
PKA agonist (25 μM sp- cAMP) rescued the frequency of mEPSC
events recorded from spinal dorsal horn neurons cocultured with
DRG (Fig.5 J and K), indicating that PKA is downstream of smCa
signals. Altogether, PKA plays an essential role in smCa- triggered
continuous neurotransmission in DRG neurons.
TRPA1
vesicle
release
Hyperalgesia
DRG
PKA
Action Potential
independent
TRPA1 smC
ac
hronic pain
spontaneous
[Ca2+]einflux
PKA
continuous
neurotransmission
Spinal cord
Ca2+
Fig. 7. Cartoon illustration showing that the AP- independent continuous
neurotransmission regulates hyperalgesia behavior. Spontaneous Ca2+ inux
through the TRPA1 channel forms a local microdomain Ca2+ signal (smCa)
independent of spontaneous APs, which continuously trigger the spontaneous
vesicle release via PKA. The smCa- mediated continuous neurotransmission
increased remarkably in chronic pain and may serve to regulate hyperalgesia
behaviors invivo. The image of spinal cord structure was modied from D.
Michael McKeough’s work
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PNAS 2025 Vol. 122 No. 3 e2406741122 https://doi.org/10.1073/pnas.2406741122 9 of 11
SmCa- Mediated Continuous Neurotransmission Regulates
Chronic Pain Behaviors. To study the physiological relevance
of smCa- mediated spontaneous vesicle release in DRG neurons,
we took advantage of the Complete Freund’s adjuvant (CFA)-
induced chronic inammatory pain model (Fig.6 A and B) (25).
Intriguingly, the ring rate and the number of active regions of
smCa activities in DRG neurons from CFA- inamed WT mice
were signicantly higher than those from saline- control mice
(Fig.6 C and D and Movies S7 and S8). However, the eect of CFA
was diminished in TRPA1−/− mice (Fig.6D and Movies S9 and
S10). Consistent with smCa activities, the spontaneous release of
NPY- pHluorin- labeled vesicles was increased remarkably in DRG
neurons from CFA- injected WT mice than that receiving saline
injection (Fig.6 E and F and Movies S11 and S12). Again, the
spontaneous release of TRPA1−/− DRG neurons was insensitive
to CFA inammation (Fig.6F and Movies S13 and S14). ese
data indicate that smCa- mediated continuous neurotransmission
is elevated in CFA- induced chronic pain. Next, we performed
hot- plate and mechanical Von Frey behavior tests for CFA- and
saline- injected WT or TRPA1−/− mice. As expected, compared
with the saline control group, CFA- inamed WT mice exhibited
signicant pain algesia with shorter latencies of paw licking on
the hot plate and lower mechanical pain threshold (Fig.6 G and
I). Importantly, the CFA- induced pain algesia was alleviated in
TRPA1−/− mice (Fig. 6 G and I), or in WT mice preinjected
with dierent doses of PKA inhibitor H89 (Fig.6 H and J).
Consistent with the blockade eects on smCa activities (Fig.1
F–M), local application of BAPTA- AM, but not EGTA- AM,
eectively alleviated the CFA- induced hyperalgesia in WT mice
(SIAppendix, Fig.S8 A and B). Furthermore, BAPTA- AM, but
not EGTA- AM, increased the threshold of mechanical pain in
CFA- treated WT mice to a similar level as that of the CFA-
inamed TRPA1−/− mice (SIAppendix, Fig.S8 C–E). us, smCa-
mediated continuous neurotransmission may play an essential
role in chronic pain.
Discussion
Chronic pain is a multifaceted and unpleasant condition that
aects more than 30% of individuals worldwide ( 1 , 2 ). In the
current study, we found that the primary sensory DRG neurons
exhibit frequent AP-independent smCa activities, which trigger
continuous neurotransmission via the cAMP-PKA signaling path-
way. Importantly, the smCa-mediated continuous neurotransmis-
sion increased dramatically in chronic pain and may play an
important role in hyperalgesia ( Fig. 7 ).
One of the major ndings of the current work is the hotspots
of AP-independent smCa activities in the somatic and axonal
regions of primary sensory DRG neurons, supported by the fol-
lowing evidence: i) By using TIRF live-imaging, we observed
frequent local Ca2+ transients in both the somata and axons of
DRG neurons without excitatory stimuli ( Fig. 1 A –E , SI Appendix,
Fig. S1 A and B , and Movies S1–S4 ). ii) e spontaneous local
Ca2+ transients were largely blocked by the fast Ca2+ -chelator
BAPTA-AM, but not the slow Ca2+ -chelator EGTA-AM ( Fig. 1
F –M ), indicating the local Ca2+ transients to be microdomain
Ca2+ signals. iii) e microdomain Ca2+ signal is independent of
membrane potential or the ring of APs ( Fig. 2 D and E , Left and
SI Appendix, Fig. S2 C–F ). iv) e smCa signals are critically
dependent on extracellular Ca2+ inux ( Fig. 2 A –C and
SI Appendix, Fig. S1 A–E ). v) e nonselective TRP channel
blocker, RR almost eliminated the smCa signals in DRG neurons
( Fig. 2F and SI Appendix, Fig. S2 I and J ). vi) e TRPA1 channel
antagonists, A967079 and HC030031, reduced the smCa signals
in both the somata and axons of DRG neurons signicantly
( Fig. 2G and SI Appendix, Fig. S3 ). vii) e smCa events were
diminished in TRPA1−/− DRG neurons ( Fig. 2I and SI Appendix,
Fig. S4 ) and increased in the DRG neurons with TRPA1 overex-
pression ( Fig. 2J and SI Appendix, Fig. S5 ). Taken together, the
spontaneous opening of TRPA1 channels mediates smCa activities
in DRG neurons.
TRPA1 is one of the TRP channels expressed in the nociceptive
DRG neurons and involved in multiple sensations, including
cold, mechanical, and itchy sensations ( 26 – 28 ). In addition,
TRPA1 is activated by a broad range of reactive chemicals and
inammatory agents (protons, fatty acid derivatives, cytokines,
chemokines, etc.), and thus plays an important role in analgesia
( 29 , 30 ). Our current study demonstrates that the spontaneous
activation of TRPA1 is the primary source of the frequent smCa
activities in DRG neurons. Interestingly, TRPA1 may not be the
only channel responsible for smCa signals because we observed
smCa activities in nearly all types of DRG neurons, not just the
small-sized DRG neurons where the TRPA1 channel is com-
monly found ( 31 ). Our present study focused on small-sized
DRG neurons because of their crucial roles in pain sensation.
However, there was still a small number of smCa activities
remaining in the small-sized DRG neurons incubated with
TRPA1 antagonist or from TRPA1−/− mice ( Fig. 2 G and I and
SI Appendix, Figs. S3 and S4 ). Since the nonselective TRP chan-
nel antagonist, RR almost eliminated the smCa events ( Fig. 2F ),
the remaining TRPA1-independent smCa activities might be
contributed by some other TRP channels. e origin and func-
tion of the remaining smCa signals in DRG neurons need further
investigation. In addition, our current work is mainly performed
in cultured neurons, and we shall continue to explore the smCa
activities in more physiological conditions in the future.
As an important second messenger, local Ca2+ signals are
involved in multiple cellular procedures, including excitation–
contraction coupling ( 32 ), gene expression ( 33 ), cell migration
( 34 ), and neurotransmission ( 21 , 35 ). Our data demonstrate that
TRPA1-mediated smCa activities trigger spontaneous neuro-
transmission in DRG neurons, including somatic and axonal
release of neurotransmitters or neuromodulators observed with
real-time TIRF imaging of the vesicles labeled with Spy-pHluorin
or NPY-pHluorin ( Fig. 3 and Movies S5 and S6 ), and glutamate
release from the synapses formed between DRG and dorsal horn
neurons detected by mEPSC recordings ( Fig. 4 ). e crucial roles
of Ca
2+
in AP-triggered neurotransmission have been well estab-
lished that VGCCs-mediated Ca2+ inux triggers vesicle release
via the Ca2+ sensor of synaptotagmin ( 36 , 37 ). On the other
hand, Ca2+ also contributes essentially to spontaneous neuro-
transmission, but the Ca2+ -sensor for spontaneous neurotrans-
mission is still controversial ( 38 , 39 ). PKA has been shown to
enhance spontaneous neurotransmitter release via the phospho-
rylation of complexin ( 24 ). PKA also plays an important role in
regulating the releasable vesicle pool ( 40 ), which in another way
facilitates spontaneous neurotransmitter release. Importantly,
two adenylyl cyclases (AC1 and AC8) are known to be activated
by Ca2+ /calmodulin ( 41 ), rendering PKA a potent candidate
involved in Ca2+ -triggered spontaneous neurotransmission.
Indeed, our data showed that the specic antagonist of PKA
(H89) substantially reduced the spontaneous release of
NPY-pHluorin labeled vesicles ( Fig. 5 A –C ) and mEPSC events
from dorsal horn neurons cocultured with DRG neurons ( Fig. 5
D and E ). In addition, sp-cAMP, an activator of PKA restored
the mEPSC events after inhibiting the smCa signals with TRPA1
antagonist ( Fig. 5 J and K ), indicating that PKA is downstream
of smCa. However, we could not fully exclude the potential
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10 of 11 https://doi.org/10.1073/pnas.2406741122 pnas.org
long-term eect of PKA on smCa, which might have a systematic
impact on the plasticity changes at molecular, structural, and
functional levels. Taken together, PKA plays an essential role in
smCa-mediated continuous neurotransmission from DRG neu-
rons to the central spinal cord.
As the primary sensory neurons, DRG neurons play a pivotal role
in the development and maintenance of chronic pain. In dorsal root
ganglia and dorsal horn where pain signal is transmitted from DRG
neurons to the central nervous system, the extracellular concentration
of dierent neurotransmitters (glutamate, ATP, etc.) and neuromod-
ulators (Substance P, CGRP, NPY, etc.) are accumulated in chronic
pain, which contributes essentially to pain behaviors ( 10 – 15 ). e
elevated concentration of these neurotransmitters and neuromodu-
lators is thought to be caused by the increased ring rate of sponta-
neous APs of DRG neurons in chronic pain ( 42 ). Our data, however,
oers direct evidence that spontaneous vesicle release from both the
somata and the axons of DRG neurons is independent of APs
(SI Appendix, Fig. S6E ). Instead, TRPA1-mediated smCa activities
and the downstream PKA signaling pathway are essential for the
AP-independent spontaneous neurotransmission in DRG neurons
( Figs. 3 – 5 ). Importantly, the smCa activity and its triggered sponta-
neous neurotransmission were increased signicantly in animals
experiencing inammatory pain ( Fig. 6 C –F and Movies S7–S14 ),
and the CFA-induced pain hyperalgesia was alleviated by TRPA1−/−
or PKA antagonist, shown to inhibit smCa-mediated spontaneous
neurotransmission ( Fig. 6 G –J ). It is not surprising that TRPA1 and
PKA are involved in CFA-induced hyperalgesia because they are well
studied in previous literature ( 43 , 44 ). TRPA1 is known to be
expressed in nociceptive DRG neurons and involved in both physi-
ological noxious-stimuli sensations and chronic pain hyperalgesia
( 26 – 28 ). For PKA, it has been reported that continuous activation
of the cAMP-PKA pathway maintains the hyperexcitability of sen-
sory neurons and behavioral hyperalgesia ( 45 , 46 ), and the antago-
nists targeting the cAMP-PKA pathway greatly suppress thermal and
mechanical hyperalgesia ( 47 – 49 ). Additionally, we found that
BAPTA-AM, but not EGTA-AM, signicantly alleviated the
CFA-induced hyperalgesia in vivo (SI Appendix, Fig. S8 ), suggesting
the specic role of TRPA1-mediated smCa in chronic pain. us,
our study reveals a potential role of AP-independent continuous
neurotransmission from DRG neurons in hyperalgesia, which may
provide a unique target for the treatment of chronic pain in the future.
Materials and Methods
The use and care of animals were approved and directed by the Institutional
Animal Care and Use Committee of Xi’an Jiaotong University and Peking
University and the Association for Assessment and Accreditation of Laboratory
Animal Care. High- resolution live- cell TIRF imaging was adopted to observe
smCa transients and quantal vesicle release in cultured sensory DRG neurons
from juvenile or adult rodents. Patch- Clamp was adopted to record mEPSCs
in cocultured DRG- spinal dorsal horn synapses or fresh spinal cord slices with
DRG afferent fibers from adult male rats. Hot Plate and Von Frey behavior
tests were adopted to record the pain hyperalgesia in the CFA- inflamed mice
model. For more details about the materials and methods, please see our
SIAppendix file.
Data, Materials, and Software Availability.
All study data are included in the
article and/or supporting information.
ACKNOWLEDGMENTS. We thank Prof. Zhuan Zhou at Peking University for
providing the TRPA1−/− and TRPV1−/− mice. This work was supported by the
National Natural Science Foundation of China (32400650, 82201404, 32171233,
32300819), the China Postdoctoral Science Foundation (2020M680211,
2021T140014, GZC20232111), the Natural Science Foundation of Shaanxi
Province of China (2024JC- YBMS- 141, 2023- ZDLSF- 23, 2024JC- YBMS- 146),
the Fund of Shaanxi Province of China (2023SYJ09), and the Shaanxi Postdoc
Funding (2023BSHTBZZ15, 2023BSHYDZZ39).
Author aliations: aDepartment of Neurology, the Second Aliated Hospital,
Neuroscience Research Center, Key Laboratory of Biomedical Information Engineering
of Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University,
Xi’an 710000, China; bNeurological Department of Tongji Hospital, School of Medicine,
Tongji University, Shanghai 200333, China; cKey Laboratory of Medical Electrophysiology
of Ministry of Education, Collaborative Innovation Center for Prevention and Treatment
of Cardiovascular Disease, Institute of Cardiovascular Research, Southwest Medical
University, Luzhou 646000, China; and dSolomon H. Snyder Department of Neuroscience,
Johns Hopkins University School of Medicine, Baltimore, MD 21205
Author contributions: Z.Z., C.W., Z.C., and R.H. designed research; Z.Z., J.Y., J.H., R.W., X.D.,
Y.C., H.X., and R.H. performed research; Z.Z., J.Y., J.H., and R.H. analyzed data; and Z.Z.,
C.W., Z.C., and R.H. wrote the paper.
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