June 2022

Medical Mass Spectrometry Vol. 6 No. 1

Research Paper

Endocannabinoid 2-AG inhibits the release of dopamine from PC12 cells
1

1

1

1

Hiroshi Ochi *, Yukari Hirata , Makoto Hamajima , Shuji Kozawa ,2,31

Kazuo Igarashi , Ichiro Isobe
1

Department of Legal Medicine, Fujita Health University School of Medicine,1‒98 Dengakugakubo, Kutsukake-cho, Toyoake, Aichi 470‒1192, Japan
2
Association of Medicinal Analysis, 5‒1‒522‒417 Koyochonaka, Higashinada-ku,
Kobe, Hyogo 658‒0032, Japan3
Department of Legal Medicine, Kinki University School of Medicine,377‒2 Oonohigashi, Osakasayama, Osaka 589‒8511, Japan
Abstract

The endocannabinoid system (ECS) is known to be involved in the retrograde suppression of signal transduc-tion at synapse. Although there are several reports investigating the eﬀect of ECS on dopamine (DA) signaling, consistent
results have not been reported. Therefore, in this study, PC12 cells diﬀerentiated with nerve growth factor were used asmodel cells for dopaminergic neuron, and the true eﬀects of endocannabinoid on DA release in these cells were accurately
evaluated using mass spectrometry. The induction of DA release by hexanal was signiﬁcantly suppressed by co-treatment
with endocannabinoid 2-arachidonylglycerol (2-AG). In addition, it was suggested that the changes in intracellular calcium
kinetics mediated by cannabinoid receptors were involved in the suppression of DA release by co-treatment with 2-AG. We
have also shown in previous studies that DA enhances the function of the ECS in glial cells. These results suggest that glial
ECS may act as a suppression system for DA signaling in situations where DA signaling is abnormally enhanced in the central nervous system.
Key words: endocannabinoid system, 2-arachidonylglycerol, dopamine, PC12 cells, calcium kinetic

Introduction

these cannabinoid receptors and endocannabinoids, the system composed of enzymes involved in the synthesis and deg-After the 1990s, cannabinoid receptors were identiﬁed asreceptors for tetrahydrocannabinol, the major psychotropic
component of cannabis sativa. Currently, cannabinoid recep-

radation of endocannabinoids (N-acyl phosphoatidylethanol-

tor type 1 and type 2 (CB1 and CB2) are known as the major
1,2)cannabinoid receptors . Thereafter, arachidonylethanol-

diacylglycerol lipase alpha, fatty acid amide hydrolase andmonoglyceride lipase) is known as the endocannabinoid sys-amide and 2-arachidonylglycerol (2-AG) have been identiﬁedtem (ECS) . This system has been shown to be involved in

amide

D,

phospholipase

C

beta

4,

6)

3‒5)

as the endogenous ligand of these receptors

phospholipase

7‒13). In addition to

the regulation of various physiological functions.

The central nervous system (CNS) is composed of neurons* Corresponding author

and glial cells. Neurons play a central role in signal transduc-

Hiroshi Ochition in the CNS. When a stimulus above the threshold is

Department of Legal Medicine, Fujita Health University
School of Medicine, 1‒98 Dengakugakubo, Kutsukake-cho,applied to a neuron, depolarization occurs and an action
potential is generated. The action potential conducts on the

Toyoake, Aichi 470‒1192, Japan
Tel: ＋81‒562‒93‒2438, Fax: ＋81‒562‒92‒4580

axon. At the axon terminal, synapse is formed with otherneuron, and signal transduction between neurons is carriedout by converting electrical signals into chemical neurotrans-

E-mail: hochi@fujita-hu.ac.jp

Received: July 2, 2021. Accepted: August 18, 2021.

mitters. The neurotransmitters released from presynaptic terminals induce depolarization or hyperpolarization of postsyn-

Epub September 24, 2021.
DOI: 10.24508/mms.2022.06.001
27

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Medical Mass Spectrometry Vol. 6 No. 1

aptic neuron. These conﬂicting signal transductions interact

try, which is only an indirect evidence. In order to improvethese problems, we used PC12 cells as the model of dopa-

with each other to regulate neuronal activity. Glial cells, theother cells that constitute the CNS, are classiﬁed into oligodendrocytes, microglia, and astrocytes. These glial cells pro-minergic neuron in this study. In addition, mass spectrometry was used to directly evaluate the eﬀect of 2-AG on thevide environmental maintenance and metabolic support in
the CNS for neuronal survival and function. Oligodendro-

release of endogenous DA in these cells. Furthermore, we
also investigated the eﬀect of 2-AG on intracellular calcium

cytes are involved in axon signaling and microglia areion behavior because it is known that intracellular calcium

involved in the immune response in the CNS. Astrocytes are

dynamics are deeply involved in not only the release of

involved in the regulation of signal transduction in the CNS

neurotransmitters but the suppression of synaptic signal

14)

by forming tripartite synapses with neurons .

transduction by ECS. We considered that our experimental

The ECS is involved in the retrograde suppression of sig-model could reveal the true eﬀect of ECS on DA signaling.

nal transduction at synapses. This mechanism begins withthe release of endocannabinoids from postsynaptic neuron

Materials and Methods

into the synaptic cleft. The endocannabinoids suppress theelevation of intracellular calcium ions in presynaptic neu-

Reagents and chemicals

rons through binding to cannabinoid receptors. As a result,(Darmstadt, Germany), and listed as follows: nerve growth
factor (NGF), hexanal, 2-AG, isoproterenol, triﬂuoroacetic

All reagents and chemicals were purchased from Merck

the release of neurotransmitters from presynaptic neuron issuppressed. Among such inhibition of synaptic transduction
with ECS, the suppression of gamma-aminobutyric acidacid (TFA), acetonitrile (MeCN), N,O-bis(trimetylsilyl)tri-(GABA) release at inhibitory synapses is known as depolar15,16), and theization-induced suppression of inhibition (DSI)

calcium ionophore (A23187) and ethyleneglycol bis

suppression of glutamic acid (Glu) release at excitatory
synapses is known as depolarization-induced suppression of(ab23703) and CB2 (ab45942)) were purchased fromﬂuoroacetamide＋trimetylchlorosilane(BSTFA＋TMCS),

tetraacetic acid (EGTA). The primary antibodies (CB1
Abcam plc (Cambridge, UK) and secondary antibody was

17)

excitation (DSE) . Such DSI and DSE have been observed
18)
in many brain regions . In addition to GABA or Glu sig-

purchased from Cell Signaling Technology (MA, USA).

naling, it is known that various other neurotransmitter sig-

Cell culture and treatment

19‒23)

naling are regulated by ECS

The PC12 rat pheochromocytoma cell line was pur-.

In our previous study, we investigated the relationshipchased from American Type Culture Collection (VA, USA).

between dopamine (DA) signaling and the ECS and foundthat long-term high concentrations of DA induced theCells were cultured in Dulbecco s Modiﬁed Eagle s
Medium (DMEM, Merck, Darmstadt, Germany) supplemented with 10% v/v heat-inactivated horse serum (Merck,

2-AG-productivity in glial cells . From these facts, we
24)

DA signaling when synaptic DA signaling is abnormally

Darmstadt, Germany) and 5% v/v heat-inactivated fetal
bovine serum (Merck, Darmstadt, Germany) in a humidi-

enhanced. Although there are several reports investigatingﬁed incubator at 37°
C with an atmosphere of 5% CO2.

the eﬀect of ECS on DA signaling, consistent results have25‒29). It seems that the reason why consisnot been reported

Cells were reseeded in the 6 well plates (for western blot
assay and quantiﬁcation of DA) or 96 well plates (for mea-

tent results have not been reported is the experimental mod-surement of intracellular Ca ) at a density 4×10 cells/cm .

els and methods are not suitable. First, slice culture of the

To investigate the morphological change and expression

brain was used in these previous reports and it is possible

change of cannabinoid receptors due to NGF treatment,that they detected the results of the interaction of multiple
types of cells present there. Second, these results may diﬀer

PC12 cells were treated with 0 ng/mL NGF or 100 ng/mL
NGF and designated as NGF(−)-PC12 or NGF(＋)-PC12,

from the real behavior of endogenous DA release, because

respectively. The treatment with NGF was performed for 7

they were investigating the eﬀects of endocannabinoids on
the release of radiolabeled-DA. In addition, other reports

days, and cell morphological observation and sampling ofspeculate that glial ECS acts as an inhibitory mechanism of

2＋

4

2

total protein were performed every day from day 1 to day 7.
To investigate the expression levels of CB1 and CB2 canna-

have detected the eﬀect of endocannabinoid on the releaseof DA by electrophysiological techniques using voltamme-binoid receptors, these cells were collected by using cell lysis
28June 2022

Medical Mass Spectrometry Vol. 6 No. 1

buﬀer (Cell Signaling Technology, MA, USA). For quantiﬁ-

jector (Shimadzu, Kyoto, Japan). Analysis was performedin positive electron ionization mode. The GC/MS condi-

cation of DA, reseeded cells were cultured with NGF for 4
days. The DA release from NGF(＋)-PC12 was induced bytions were as follows: column, DB-5MS (30 m×0.25 mm,
i.d. 0.25μm, Agilent, CA, USA). The column oven tem-treatment with 10 mM hexanal. In addition, to assess theeﬀect of 2-AG on DA release from NGF(＋)-PC12 with hex-

perature was programed to be held at 120°
C for 3 min,anal treatment, these cells were co-treated with hexanal and
2-AG. Five minutes after, culture supernatant and cells

increased at 10°
C/min to 180°
C, at 5°
C/min to 190°
C, andwere collected for DA analysis and protein quantiﬁcation,interface, and ion source temperature, 270°
C, 260°C andrespectively. For measurement of change of intracellular200°
C, respectively; carrier gas and ﬂow rate, helium at

at 15°
C/min to 280°
C, then held for 5 min. The injector,

2＋

30)

Ca , reseeded cells were cultured with NGF for 4 days andloaded with Fluo4-AM (DOJINDO, Kumamoto, Japan).

1 mL/min . MS detection was carried out in selected ion
monitoring (SIM) mode using m/z values of 280 for
DA-TMS, m/z values of 355 for I.S.-TMS. The limit ofdetection and limit of quantiﬁcation for DA-TMS were
2 ng/mL and 10 ng/mL, respectively. Six-point calibration

Western blot assay
The concentrations of total protein were determined by
the Quant-iT Protein Assay kit and Qubit ﬂuorometer

curves were constructed for DA-TMS in the concentration
2
range 100‒600 ng/mL (r ＝0.999). In addition, the ﬁnal con-

(Thermo Fisher Scientiﬁc, MA, USA). Twenty microgramsof total proteins were separated by sodium dodecyl sul-

centration of DA was standardized by total protein concen-fate-polyacrylamide gel electrophoresis (BIO RAD, CA,
USA) and transferred to the polyvinylidene ﬂuoride mem-tration of cells derived from the same well.

brane (Merck, Darmstadt, Germany). The membrane was

Measurement of intracellular Ca with Fluo4

incubated with the primary antibody (1 : 1000 for CB1 andTo detect the change in [Ca ]i treated with hexanal and
2-AG, the Fluo4-AM dye, a calcium indicator, was loaded

2＋2＋

1 : 500 for CB2) at room temperature for 1 h and with
anti-rabbit secondary antibody (1 : 10000 for CB1 andinto our dopaminergic model cells (NGF (＋)-PC12). The
ﬂuorescence intensity of Fluo4-loaded cells was measured

1 : 5000 for CB2) at room temperature for 1 h. The ECL

by using ARVO X5 multilabel reader (Parkin Elmer, MA,
USA). After baseline was collected at an emission wave-

select (Cytiva, Tokyo, Japan) was used to determine theexpression levels of the proteins. Immunoreactive bands
Rasband, national Institutes of Health).

length of 535 nm and excitation wavelength of 485 nm for
20 s, hexanal and 2-AG were added to culture supernatant.

Solid phase extraction and derivatization of DA

The change of ﬂuorescence intensity of Fluo4 was detected2＋
for 200 s at the same condition. The intracellular Ca con-were analysed by densitometry using ImageJ 1.48 v (Weyne2＋2＋centration ([Ca ]i) was calculated using equation : [Ca ]i＝

The DA in collected culture supernatants was extractedusing Bond Elut PBA (Agilent, CA, USA). The cartridge
was conditioned with MeCN, 1% TFA-MeCN and 50 mM

Kd×{(F−Fmin)/(Fmax−F)}. Kd represents the dissociation
2＋
constant for Ca -bound Fluo4 and is 345 nM. F represents

phosphate buﬀer (pH10). After loading the sample (buﬀered
to pH8.5), cartridge was washed with 10 mM phosphate buf-

the ﬂuorescence of experimental samples. Fmin represents
2＋
the minimum ﬂuorescence of Ca -bound Fluo4 by addingfer (pH8.5)＋5% MeCN and 0.1% TFA-MeCN. The DA waseluted from the cartridge with 1% TFA-MeCN. Isoproterenol

2＋
EGTA. Fmax represents the maximum ﬂuorescence of Ca -bound Fluo4 by adding A23187.

was added as an internal standard (I.S.) to eluted solution.
The eluted solution was evaporated, and the residue was deri-

Statistical analysis

vatized with BSTFA＋TMCS for 1 h at 60°
C. The trimethylsilyl (TMS) derivatized samples were analysed by gas chroma-

All data were shown as the mean±standard error (SE). A
one way analysis of variance followed by Tukey‒Kramer

tography/mass spectrometry (GC/MS).

method was used to compare the signiﬁcance of diﬀerence
among all groups. Results were considered signiﬁcant at *p

Quantiﬁcation of DA by GC/MS＜0.05 and **p＜0.01.

GC/MS analysis was performed on GCMS-QP2010 (Shimadzu, Kyoto, Japan) equipped with an AOC-20i auto-in29

June 2022

Medical Mass Spectrometry Vol. 6 No. 1

Results

reached the highest value at 10 s after the addition of the

Change of cellular morphology and expression stateof cannabinoid receptors in PC12 cells by NGF
treatment

agent, and these values were shown below: 4176±1086 nM
for hexanal＋1 nM 2-AG, 2977±288 nM for hexanal＋10 nM

In NGF(−)-PC12, cell proliferation was observed, but no2-AG, and 1725±183 nM for hexanal＋100 nM 2-AG, (Fig.
2＋3). The [Ca ]i were markidly increased by hexanal treat-signiﬁcant change in its morphology was observed (Fig.
1A). On the other hand, in NGF(＋)-PC12, extension of neu-

ment (P＜0.01, compared with cont.), and this increase wassigniﬁcantly suppressed in dose-dependent manner ofrite began to be observed after day 2 in addition to cell pro-co-treated 2-AG (P＜0.01, compared with 10 mM hexanal＋
0 nM 2-AG).

liferation, and these neurites became a network structurewith each passing day (Fig. 1B). The expression states of
the cannabinoid receptors (CB1 or CB2) in these
NGF(−)-PC12 or NGF(＋)-PC12 were conﬁrmed by west-

Discussion

ern blotting method. Although the expression states of CB1
in NGF(−)-PC12 were slightly up-regulated with the prog-

1976 from rat adrenal pheochromocytoma . These cells
have the ability to release some neurotransmitters (nor-ress of culture, the expression states of CB1 in
NGF(＋)-PC12 were highest on day 4, and this expression

adrenaline, catecholamines, and DA) by vesicles. In addi-

PC12 cells were established by Greene and Tischler in
31)tion, since this cell diﬀerentiates into a sympathetic nerve
in response to NGF, it is often used as a model cell for exo-

level was signiﬁcantly higher than that in NGF(−)-PC12

32)(Fig. 1C). On the other hand, the expression states of CB2
were not signiﬁcantly changed in either NGF(−)-PC12 or

cytosis studies of such neurotransmitters . CB1 and CB2

NGF(＋)-PC12 (Fig. 1D). We used the NGF(＋)-PC12 onof these receptors is distinctly diﬀerent: CB1 is expressed

day 4 as the model of dopaminergic neuron in subsequent

mainly in the nervous system, whereas CB2 is expressed

experiments because the neurites are suﬃciently extended

mainly in the peripheral immune system tissue . It was

and CB1 is the most highly expressed.

considered that the sensitivity to endocannabinoid may

are known as major cannabinoid receptors. The distribution

33)

change by diﬀerentiation with NGF. Therefore, to use PC12

Eﬀect of 2-AG on DA release

In Fig. 2A, chromatograms from DA-TMS and I.S.-TMS

cells as a model of dopaminergic neuron in this study, we
ﬁrst conﬁrmed the expression state of cannabinoid recep-

in collected sample were shown. The concentrations oftors (CB1 and CB2) by NGF-treated PC12. As a result, theextracellular DA after the treatment were as shown below:
0.17±0.01 ng/mg protein for 0 mM hexanal＋0 nM 2-AG,

expression level of CB1 increased due to the diﬀerentiation

155.29±31.28 ng/mg protein for 10 mM hexanal＋0 nM
2-AG, 73.85±20.15 ng/mg protein for 10 mM hexanal＋

change was observed in the expression level of CB2. These
increase in expression level of CB1 associated with the dif-

1 nM 2-AG, 41.66±13.94 ng/mg protein for 10 mM hexanal＋10 nM 2-AG, and 48.66±7.29 ng/mg protein for 10 mM

ferentiation of PC12 cells into neuron-like cell by NGF

of PC12 cells by NGF treatment, although no signiﬁcanttreatment suggest not only morphological change butchange of cell properties into neuron-like cell. Based on

hexanal＋100 nM 2-AG (Fig. 2B). The DA release from
NGF(＋)-PC12 were markedly increased by hexanal treat-thesefacts, PC12 cells diﬀerentiated by NGF(NGF(＋)-PC12) were used as the model of dopaminergic

ment, and these induction of DA release were signiﬁcantly
decreased in a dose-dependent manner of co-treated 2-AG.

neuron in the subsequent our studies.
Generally, the release of neurotransmitters from presyn-

Similar results were reproduced by multiple independent
experiments.

aptic terminals begins with the inﬂux of calcium ions into
the cell through voltage-gated calcium channels after the2＋
Eﬀect of 2-AG on change of [Ca ]iaction potential reaches the presynaptic terminal. The ele-

In the control sample (cont.), no signiﬁcant change wasvation of intracellular calcium ions causes synaptic vesicles

2＋

observed in [Ca ]i (Fig. 3). When the cells were treated withcontaining neurotransmitters to fuse with the presynaptic

2＋

10 mM hexanal alone, [Ca ]i reached the highest value

membrane and release neurotransmitters into the synaptic(16505±2287 nM) at 10 s after the addition of the agent (Fig.
2＋3). The [Ca ]i when co-treated with hexanal and 2-AG also

cleft. In other words, the progress of signal transduction at
2＋

synapses is triggered by an increase in [Ca ]i. In this study,30June 2022

Fig. 1.

Medical Mass Spectrometry Vol. 6 No. 1

Change of cellular morphology and expression state of cannabinoid receptors in PC12 cells by NGF treatment.

(A) and (B) show the cellular morphology of PC12 cells treated with 0 ng/mL NGF (NGF(−)-PC12) and 100 ng/mL NGF (NGF(＋)-PC12), respectively. (C) and (D) show the result of western blot of CB1 and CB2, respectively. White bars refer to NGF(−)-PC12 and black bars refer to NGF(＋)-PC12. **P＜0.01, compared with NGF(−)-PC12.

31

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Medical Mass Spectrometry Vol. 6 No. 1

Fig. 2.

Eﬀect of 2-AG on DA release of NGF(＋)-PC12 cells.

Fig. 3.

Eﬀect of 2-AG on change of [Ca ]i.

(A) The SIM chromatograms of DA-TMS and I.S.-TMS in collected sample. (B) DA released into culture supernatant by co-treatedwith hexanal and 2-AG. All results were showed as mean±SE (n＝3). *P＜0.05 and **P＜0.01, compared with 0 mM hexanal＋0
nM 2-AG.

2＋

Intracellular Ca kinetics in NGF(＋)-PC12 cells co-treated with hexanal and 2-AG were measured using Fluo4. All results were expressed as the mean±SE from three independent preparations.
2＋

we used PC12 cells as the model of dopaminergic neuron toinvestigate the eﬀect of 2-AG on the release of DA inherent

by the treatment of hexanal, and these induction of releaseof DA by hexanal was signiﬁcantly suppressed in a dose-de-in these cells. Hexanal was used to induce the release of
DA from these cells. In addition, the ECS is involved in ret-pendent manner of 2-AG. Hexanal is straight-chain ali-rograde suppression of signal transduction at synapses.
Therefore, we examined whether endocannabinoid 2-AG

induce the release of DA from PC12 cells by increasing theacts suppressively on DA release from the model of dopaminergic neuron (NGF(＋)-PC12). As a result, it was clari-hexanal is a good experimental model of the physiologicalphatic aldehyde having 6 carbon atoms, and is known to2＋ 34)[Ca ]i . It seems that the induction of release of DA byprocess of release of neurotransmitters at synapses. Fromthese facts, we speculate that 2-AG also suppresses theﬁed that the extracellular DA level was markedly increased32June 2022

Fig. 4.

Medical Mass Spectrometry Vol. 6 No. 1

Schema of inhibitory mechanism of DA release by glial endocannabinoid 2-AG.

2＋
Generally, DA release from synaptic terminal of dopaminergic neuron is promoted in a [Ca ]i-depending manner. DA release fromDA neurons is also abnormally enhanced by the action of addictive drugs such as stimulants. These enhancements in DA signaling
are detected by astrocytes coexisting at synapses. The productivity of 2-AG in astrocytes are induced due to the changes in the expression of factors involved in the turnover of 2-AG in these cells. The 2-AG released from astrocytes into the synaptic cleft binds
to cannabinoid receptors exist at presynaptic terminals. The action of Gi/o protein coupled to these receptors suppresses the increase2＋
in [Ca ]i, resulting in the suppression of DA release from presynaptic terminals. In other words, astrocytic ECS acts as the inhibitory mechanism for DA signaling.

We have been investigating the eﬀects of glial endocan-

release of DA from dopaminergic neuron in vivo.
The retrograde suppression of signal transduction at synapses by ECS is initiated by the binding of endocannabi-

nabinoid systems on DA signaling. In our previous study,
we revealed that the long-term exposure of high-concentra-

noids to cannabinoid receptors present at presynaptic termi-

tion DA induced the 2-AG-productivity in glial cells

nals. These cannabinoid receptors are classiﬁed as
G-protein-coupled receptors, and CB1 is coupled with Gi/o.

through changes in the expression state of factors involved
24)
in 2-AG turnover . A common site aﬀected by addictive

The action of Gi/o proteins through activation of thesereceptors by endocannabinoids induce not only the inhibi-

drugs is the dopaminergic neuron, which projects from thetion of voltage-gated calcium channel but the activation ofvoltage-gated and inward-rectiﬁer potassium channels. As adopaminergic neuron, also called the reward system, is35‒37)

ventral tegmental area to the nucleus accumbens. Suchdeeply involved in the formation of drug dependence. It isthought that repeated use of addictive drugs causes abnor-

result, the inﬂux of calcium ions into the cell is suppressed,
and ﬁnally the release of neurotransmitters from the presyn-

malities in CNS function due to long-term stimulation ofapse is suppressed. The calcium ion dynamics are deeply
involved in both the release of neurotransmitters at syn-the reward system, resulting in drug dependence. From theresults of our previous studies, it is suggested that the func-

apses into the synaptic cleft and the suppression of synaptic

tion of ECS in glial cells present at synapses is enhanced

signal transduction by ECS. Therefore, we investigatedwhether calcium ion dynamics was involved in the suppres-under the condition that DA at synaptic cleft is abnormallyhigh due to long-term repeated use of addictive drugs. Fur-sion of DA release by 2-AG observed in our experimental

thermore, in this study, we revealed that endocannabinoid
2-AG actually suppresses the release of DA from dopami-

2＋model. As a result, the increase in [Ca ]i by hexanal was
signiﬁcantly suppressed in dose-dependent manner of
2-AG. These facts demonstrated that our experimental sys-

nergic neuron. Thus, these results suggest that glial ECS
may act as a suppression system for DA signaling in situa-

tem suﬃciently reproduced the mechanism of suppression

tions where DA signaling is abnormally enhanced in theof signal transduction at synapses by ESC at the cultured

central nervous system (Fig. 4).

cell level.
33

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Medical Mass Spectrometry Vol. 6 No. 1

Conclusion

cannabinoid system. Biol Psychiatry 79: 516‒525, 2016.

The purpose of this study was to reveal the true eﬀect of
ECS on DA signaling by improving the problems of previ-7) Morena M, Campolongo P: The endocannabinoid system:
An emotional buﬀer in the modulation of memory function. Neurobiol Learn Mem 112: 30‒43, 2014.

ous report that investigated the eﬀect of ECS on the releaseof DA. Using PC12 cells as a model for dopaminergic neu-

8) Bellocchio L, Cervino C, Pasquali R, Pagotto U: The en-rons, we investigated the eﬀect of 2-AG on the release of

docannabinoid system and energy metabolism. J Neuro-

endogenous DA in these cells. As a result, it was clariﬁed

endocrinol 20: 850‒857, 2008.

that the release of DA under physiological conditions was
2＋
inhibited by suppressing the increase in [Ca ]i by 2-AG.

9) Wang L, Yang T, Qian W, Hou X: The role of endocanna-

Considering the results of our previous studies and thismovement sleep deprivation in rats: Regional diﬀerences.

study, it is suggested that glial ECS coexisting at synapses

Int J Mol Med 27: 119‒126, 2011.

binoids in visceral hyposensitivity induced by rapid eye

may act as a inhibitory mechanism for abnormally

10) Basu S, Dittel BN: Unraveling the complexities of canna-enhanced DA signaling. In other words, ECS may have anbinoid receptor 2 (CB2) immune regulation in health andantagonistic eﬀect on addictive drugs, and it is suggested

disease. Immunol Res 51: 26‒38, 2011.

that extrinsic activation of this system may be applicable to11) Kwok CH, Devonshire IM, Imraish A, Greenspon CM,
Lockwood S, et al: Age-dependent plasticity in endocan-

the treatment of drug addiction.

nabinoid modulation of pain processing through postnatal

Acknowledgement

development. Pain 158: 2222‒2232, 2017.
12) Fraga D, Zanoni CI, Rae GA, Parada CA, Souza GE: En-

This work was supported by JSPS KAKENHI Grant
numbers JP20K10567 and Grant-in-Aid for Research fromdogenous cannabinoids induce fever through the activation of CB1 receptors. Br J Pharmacol 157: 1494‒1501,

Fujita Health University.

2009.

Conﬂict of Interest13) Zlebnik NE, Cheer JF: Drug-induced alterations of endocannabinoid-mediated plasticity in brain reward regions. J

There are no conﬂict of interest relevant to this article.

Neurosci 36: 10230‒10238, 2016.

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