Opiate-induced Changes in Brain Adenosine Levels and Narcotic Drug Responses.
ABSTRACT We have very little information about the metabolomic changes that mediate neurobehavioral responses, including addiction. It was possible that opioid-induced metabolomic changes in brain could mediate some of the pharmacodynamic effects of opioids. To investigate this, opiate-induced brain metabolomic responses were profiled using a semi-targeted method in C57BL/6 and 129Sv1 mice, which exhibit extreme differences in their tendency to become opiate dependent. Escalating morphine doses (10-40 mg/kg) administered over a 4-day period selectively induced a two-fold decrease (p<0.00005) in adenosine abundance in the brainstem of C57BL/6 mice, which exhibited symptoms of narcotic drug dependence; but did not decrease adenosine abundance in 129Sv1 mice, which do not exhibit symptoms of dependence. Based on this finding, the effect of adenosine on dependence was investigated in genetically engineered mice with alterations in adenosine tone in the brain and in pharmacologic experiments. Morphine withdrawal behaviors were significantly diminished (P<0.0004) in genetically engineered mice with reduced adenosine tone in the brainstem, and by treatment with an adenosine receptor(1) (A(1)) agonist (2-chloro-N6-cyclopentyladenosine, 0.5 mg/kg) or an A(2a) receptor (A(2a)) antagonist (SCH 58261 1 mg/kg). These results indicate that adenosine homeostasis plays a crucial role in narcotic drug responses. Opiate-induced changes in brain adenosine levels may explain many important neurobehavioral features associated with opiate addiction and withdrawal.
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ABSTRACT: The metabolic profiles of urine and blood plasma in drug-addicted rat models based on morphine (MOR), methamphetamine (MA), and cocaine (COC)-induced conditioned place preference (CPP) were investigated. Rewarding effects induced by each drug were assessed by use of the CPP model. A mass spectrometry (MS)-based metabolomics approach was applied to urine and plasma of MOR, MA, and COC-addicted rats. In total, 57 metabolites in plasma and 70 metabolites in urine were identified by gas chromatography-MS. The metabolomics approach revealed that amounts of some metabolites, including tricarboxylic acid cycle intermediates, significantly changed in the urine of MOR-addicted rats. This result indicated that disruption of energy metabolism is deeply relevant to MOR addiction. In addition, 3-hydroxybutyric acid, L-tryptophan, cystine, and n-propylamine levels were significantly changed in the plasma of MOR-addicted rats. Lactose, spermidine, and stearic acid levels were significantly changed in the urine of MA-addicted rats. Threonine, cystine, and spermidine levels were significantly increased in the plasma of COC-addicted rats. In conclusion, differences in the metabolic profiles were suggestive of different biological states of MOR, MA, and COC addiction; these may be attributed to the different actions of the drugs on the brain reward circuitry and the resulting adaptation. In addition, the results showed possibility of predict the extent of MOR addiction by metabolic profiling. This is the first study to apply metabolomics to CPP models of drug addiction, and we demonstrated that metabolomics can be a multilateral approach to investigating the mechanism of drug addiction.Analytical and Bioanalytical Chemistry 08/2013; · 3.66 Impact Factor
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ABSTRACT: Interest in adenosine deaminase (ADA) in the context of medicine has mainly focused on its enzymatic activity. This is justified by the importance of the reaction catalyzed by ADA not only for the intracellular purine metabolism, but also for the extracellular purine metabolism as well, because of its capacity as a regulator of the concentration of extracellular adenosine that is able to activate adenosine receptors (ARs). In recent years, other important roles have been described for ADA. One of these, with special relevance in immunology, is the capacity of ADA to act as a costimulator, promoting T-cell proliferation and differentiation mainly by interacting with the differentiation cluster CD26. Another role is the ability of ADA to act as an allosteric modulator of ARs. These receptors have very general physiological implications, particularly in the neurological system where they play an important role. Thus, ADA, being a single chain protein, performs more than one function, consistent with the definition of a moonlighting protein. Although ADA has never been associated with moonlighting proteins, here we consider ADA as an example of this family of multifunctional proteins. In this review, we discuss the different roles of ADA and their pathological implications. We propose a mechanism by which some of their moonlighting functions can be coordinated. We also suggest that drugs modulating ADA properties may act as modulators of the moonlighting functions of ADA, giving them additional potential medical interest.Medicinal Research Reviews 06/2014; · 8.13 Impact Factor
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ABSTRACT: The effectiveness of O-pulse stimulation (TPS) for the reversal of O-pattern primed bursts (PB)-induced long-term potentiation (LTP) were examined at the Schaffer-collateral-CA1 pyramidal cell synapses of hippocampal slices derived from rats chronically treated with morphine (M-T). The results showed that slices derived from both control and M-T rats had normal field excitatory postsynaptic potential (fEPSP)-LTP, whereas PS-LTP in slices from M-T rats was significantly greater than that from control slices. When morphine was applied in vitro to slices derived from rats chronically treated with morphine, the augmentation of PS-LTP was not seen. TPS given 30 min after LTP induction failed to reverse the fEPSP- or PS-LTP in both groups of slices. However, TPS delivered in the presence of long-term in vitro morphine caused the PS-LTP reversal. This effect was blocked by the adenosine A1 receptor (A1R) antagonist CPX (200 nM) and furthermore was enhanced by the adenosine deaminase (ADA) inhibitor EHNA (10 μM). Interestingly, TPS given 30 min after LTP induction in the presence of EHNA (10 μM) can reverse LTP in morphine-exposed control slices in vitro. These results suggest adaptive changes in the hippocampus area CA1 in particular in adenosine system following repetitive systemic morphine. Chronic in vivo morphine increases A1R and reduces ADA activity in the hippocampus. Consequently, adenosine can accumulate because of a stimulus train-induced activity pattern in CA1 area and takes the opportunity to work as an inhibitory neuromodulator and also to enable CA1 to cope with chronic morphine. In addition, adaptive mechanisms are differentially working in the dendrite layer rather than the somatic layer of hippocampal CA1. © 2014 Wiley Periodicals, Inc.Journal of Neuroscience Research 06/2014; · 2.73 Impact Factor
OPIATE-INDUCED CHANGES IN BRAIN ADENOSINE LEVELS
AND NARCOTIC DRUG RESPONSES
M. WU,aP. SAHBAIE,bM. ZHENG,aR. LOBATO,a
D. BOISON,cJ. D. CLARKa,bAND G. PELTZa*
aDepartment of Anesthesia, Stanford University School of Medicine,
Stanford, CA 94305, United States
bVeterans Affairs Palo Alto Healthcare System, Palo Alto,
CA 94305, United States
cRobert S. Dow Neurobiology Laboratories, Legacy Research,
Portland, OR 97232, United States
Abstract—We have very little information about the meta-
bolomic changes that mediate neurobehavioral responses,
including addiction. It was possible that opioid-induced
metabolomic changes in brain could mediate some of the
pharmacodynamic effects of opioids. To investigate this,
opiate-induced brain metabolomic responses were profiled
using a semi-targeted method in C57BL/6 and 129Sv1 mice,
which exhibitextreme differences
to becomeopiate dependent.
doses(10–40 mg/kg) administered over a 4-day period selec-
tively induced a twofold decrease (p < 0.00005) in adeno-
sine abundance in the brainstem of C57BL/6 mice, which
exhibited symptoms of narcotic drug dependence; but did
not decrease adenosine abundance in 129Sv1 mice, which
do not exhibit symptoms of dependence. Based on this find-
ing, the effect of adenosine on dependence was investigated
in genetically engineered mice with alterations in adenosine
tone in the brain and in pharmacologic experiments.
Morphine withdrawal behaviors were significantly dimin-
ished (p < 0.0004) in genetically engineered mice with
reduced adenosine tone in the brainstem, and by treatment
with an adenosine receptor1(A1) agonist (2-chloro-N6-cyclo-
pentyladenosine, 0.5 mg/kg) or an A2areceptor (A2a) antago-
nist (SCH 58261, 1 mg/kg). These results indicate that
adenosine homeostasis plays a crucial role in narcotic drug
responses. Opiate-induced changes in brain adenosine
levels may explain many important neurobehavioral features
associated with opiateaddiction
? 2012 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: opiates, metabolomic analysis.
Efforts to improve the care of patients with chronic
pain conditions have led to a marked increase in the use
opioid analgesics are more commonly misused than all
other illicit drugs combined, including marijuana [reviewed
in (Dodrill et al., 2011)]. New approaches and a re-
evaluationof our current
mechanisms involved in narcotic drug addiction are
urgently needed.To develop
addressing this public health problem, we have been
analyzing a murine model of opiate dependence. Mice
can be made physically dependent upon morphine, and
inbred strains dramatically differ in the extent to which
they manifest various features of narcotic drug addiction,
which resemble those observed in humans (Liang et al.,
2006a,b; Chu et al., 2009). By analyzing these inter-strain
differences, we identified four genes affecting opioid
responses (Liang et al., 2006a,b; Smith et al., 2008),
including the Htr3a/5-HT3 serotonin receptor (Chu et al.,
2009). We also demonstrated that administration of a
commonly used 5-HT3 antagonist (ondansetron) reduced
narcotic drug withdrawal symptoms in mice and in normal
human subjects (Chu et al., 2009; Liang et al., 2011).
In addition to genetic data, metabolomic analysis can
reveal a great deal about the physiological state of a
tissue. We have very little information about metabolomic
diseases. It is likely that clinically important opiate
responses could be mediated (at least in part) by
metabolomic changes that are induced by opiates.
However, the extreme differences in physicochemical
properties make it impossible to accurately measure
changes in all cellular metabolites with a single analytic
method. Therefore, we coupled a recently developed
derivatization method (Guo and Li, 2009) with LC/MS
analysis to analyze changes in a large number of
metabolites in brainstem after opiate administration.
Dansylation increases metabolite detection sensitivity by
10–1000-fold, and improves metabolite retention and
separation on reversed phase columns. It enables
changes in many metabolites that have primary or
secondary amino or other groups, to be evaluated in an
unbiased fashion. This semi-targeted method was used
to characterize opiate-induced metabolomic changes in a
brain region that is critical for opiate responses in two
inbred mouse strains, which exhibit extreme differences
in the extent of physical dependence developing after
understanding of the
0306-4522/12 $36.00 ? 2012 IBRO. Published by Elsevier Ltd. All rights reserved.
Stanford University School of Medicine, 300 Pasteur Drive L232,
Stanford, CA 94305, United States.
E-mail address: email@example.com (G. Peltz).
Abbreviations: Adk, adenosine kinase; A1, adenosine A1receptor; A2a,
adenosine A2a receptor; LC/MS, liquid chromatography with mass
author. Address:Department of Anesthesia,
Neuroscience 228 (2013) 235–242
brainstem were analyzed, since this region has been
shown to regulate narcotic drug dependence (Gulati and
Bhargava, 1989; Costall et al., 1990; Tao et al., 1998).
administration.Metabolomicchanges in the
All experiments were performed according to protocols that were
approved by the Institutional Animal Care and Use Committee at
the Veterans Affairs Palo Alto Healthcare System. Male C57BL/
6J and 129/SvlmJ mice strains (7–8 weeks old) were obtained
from Jackson Laboratories (Bar Harbor, MA) and kept in our
facility for a minimum of 1 week prior to initiation of the
experiments. Mice with genetically engineered alterations in
adenosine kinase (Adk) expression (which are referred to as
Adk-tg and fb-Adk-def mice) were provided by Dr. Detlev
Boison (Legacy Research Institute, Portland, OR 97232) and
kept in our facility for 2 weeks prior to initiation of experiments.
All mice were kept under standard conditions with a 12-h light/
dark cycle and an ambient temperature of 22 ± 1 ?C. Animals
were allowed food and water ad libitum. All experiments were
performed using 7–8 mice per group, as determined by power
analyses using pilot data and from previous experiments.
Morphine (Sigma Chemicals, St. Louis, MO) was administered
subcutaneously to different groups of mice twice per day on an
ascending schedule: Day 1, 10 mg/kg; Day 2, 20 mg/kg; Day 3,
30 mg/kg and Day 4, 40 mg/kg. Vehicle (Saline) injections
followed the same twice-daily schedule. Nociceptive testing
procedures began approximately 18 h after the final dose of
initiated by sub-cutaneous injection of Naloxone (10 mg/kg) one
hour after the last dose of morphine on day 4 (Chu et al., 2009).
To study the effect of selective adenosine receptor agents on
withdrawal in morphine-treated mice, the A1R agonist 2-chloro-
N6-cyclopentyladenosine 0.5 mg/kg (Tocris Bioscience, Ellisville,
MI) or the A2AR selective antagonist SCH 58261 (Tocris
Bioscience, Ellisville, MI) 1 mg/kg or vehicle (saline) was injected
intra-peritoneally 15 min prior to naloxone administration.
Brain tissue preparation
Since rapid tissue isolation is critical for metabolomic analysis,
the total time for brainstem and frontal cortex isolation after
sacrificewas90 s using the
decapitation by guillotine, the skull was exposed and opened
along the sagittal and lambdoid sutures; the brain was then
transferred to a cold plate (4 ?C); the olfactory bulbs and
cerebellar hemispheres were removed, and the areas of
interest were separated before snap freezing on dry ice. The
areas of interest included the hindbrain (medulla and pons) and
midbrain (tectum and cerebral peduncle minus the cerebellum).
procedures developed by Guo and Li (2009). Fifty ml of the
polar metabolite extract in 0.1 M sodium tetraborate buffer was
combined with 50 ml of 20 mM dansyl chloride and vortexed.
The mixture was incubated at room temperature for 30 min
before addition of 50 ml of 0.5% formic acid to stop the
reaction. The supernatant of the reaction mixture was then
placed into an autosampler vial.
was performed usinga modification ofthe
All samples were analyzed on an Agilent (Santa Clara, CA)
accurate mass Q-TOF 6520 coupled with an Agilent UHPLC
infinity 1290 system. The chromatography runs were performed
using a Phenomenex (Torrance, CA) Kinetex reversed phase
C18 column (dimension 2.1 ? 100 mm, 2.6 mm particles, 100 A˚
pore size). Solvent A was HPLC water with 0.1% formic acid
and Solvent B was LC/MS grade acetonitrile with 0.1% formic
acid.A 30 min gradientat
t = 0.5 min, 5% B; t = 20.5 min, 60% B; t = 25 min, 95% B;
t = 30 min, 95% B. The column was balanced at 5% B for
5 min. All data were acquired by positive ESI (electrospray
ionization) with MassHunter acquisition software. Molecular
feature extraction on all data was performed using MassHunter
qual software. The metabolite abundance, which is a measure
of the metabolite concentration in an extract, was determined
using software that integrates the peak area for the indicated
metabolite on the extracted ion chromatogram for each sample.
To confirm the identity of adenosine, a targeted MS/MS
spectrum was acquired on the QTOF 6520 using the above
HPLC gradient and specified retention time with window of
0.6 min. The collision energy was set at 28 V, isolation width
4 m/z, MS acquisitionrate
acquisitionrateat 3 spectra/s.
abundances between samples in different groups, the signal
intensities for each metabolite was log-transformed, and a two-
sample two-tail t test was applied to the log-transformed data.
0.5 ml/minwasas follows:
at 5 spectra/s
Assessment of mechanical sensitivity
Mechanical sensitivity was assayed using nylon von Frey
filaments according to the ‘‘up-down’’ algorithm as described
previously to detect allodynia in mice (Liang et al., 2006a). In
these experiments, mice were placed on wire mesh platforms
in clear cylindrical plastic enclosures 10 cm in diameter and
40 cm in height. After acclimation, fibers of sequentially
increasing stiffness were applied approximately 1 mm lateral to
the central wound edge, pressed upward to cause a slight bend
in the fiber and left in place for 5 s. Withdrawal of the hind paw
from the fiber was scored as a response. When no response
was obtained the next stiffest fiber in the series was applied to
the same paw; if a response was obtained a less stiff fiber was
applied. Testing proceeded in this manner until 4 fibers had
been applied after the first one causing a withdrawal response.
The mechanical withdrawal threshold was estimated using a
data fitting algorithm that permitted the use of parametric
statistics for analysis (Poree et al., 1998).
Adult male Adk-tg, fb-Adk-def, and wild type (C57BL/6J) mice
(n = 3,each) weretrans-cardially
paraformaldehyde in PBS. Brains were removed and post-fixed
in the same fixative at 4 ?C for 1 day before being cut into
40 lm sagittal sectionsusing
immunohistochemical detection of ADK, we followed our
published procedures (Studer et al., 2006). Digital images of
ADK immunohistochemistry on 3,30-diaminobenzidine (DAB)
stained slices were acquired using a Zeiss AxioPlan inverted
microscope equipped with an AxioCam 1Cc1 camera (Carl
Zeiss MicroImaging Inc., Thornwood, NY).
with 0.15 M
a vibratome. Forthe
C57BL/6J mice become morphine-dependent after 4 days
of administration of increasing doses of morphine; the
morphine-dependent mice develop signs of withdrawal
236 M. Wu et al./Neuroscience 228 (2013) 235–242
within 18 h of their last morphine dose, and naloxone
administration rapidly induces substantial withdrawal
symptoms (Chu et al., 2009). To characterize opiate-
induced metabolomic changes, brainstem tissue was
prepared from C57BL/6J mice placed into 4 treatment
groups (n = 8 per group) (Fig. 1A): (1) Dependence:
tissue was harvested 1 h after the last morphine dose on
day 4; (2) Naloxone-induced withdrawal: the opiate
administered to morphine-dependent mice 1 h after their
last morphine dose on day 4, and then tissue was
harvested 10 min later; (3) Natural withdrawal: tissue
was harvested 18 h after their last morphine dose on
day 4, which is when withdrawal symptoms are maximal;
(4) Control: saline injections were administered over the
4-day period. Within the 32-brainstem tissues analyzed,
1300 metabolite peakswere
dependence, naloxone-induced withdrawal, or natural
withdrawal samples were compared to the control
samples, adenosine was the only metabolite whose
abundance was significantly altered in all 3 conditions
after morphine administration (Fig. 1B). The identity of
adenosine as the differentially reduced metabolite in all
3 conditions was confirmed using a chemical standard
and byLC/MS/MS analysis
adenosine abundance was significantly lower (two-fold,
p < 0.00005)in all3 conditions
morphine administration relative to that of control mice.
Thus, opiate exposure induces a 50% decrease in
(10 mg/kg s.c.)was
brainstem adenosine abundance. This represents a very
significant metabolomic response, since adenosine is an
important neuromodulator whose intracellular and extra-
cellular concentration isvery
transporters and by enzymes regulating adenosine
metabolism [reviewed in (Boison, 2006)]. Adenosine
abundance was significantly decreased during the period
maintained during the 18-h period of narcotic drug
abstinence when these mice manifest maximal signs of
dependence after 4 days of morphine administration (Chu
et al., 2009), metabolomic changes in brainstem tissues
in these mice were also examined during the dependence
and naloxone-induced withdrawal states. In contrast to
C57BL/6J mice, no metabolite exhibited a significant
changein 129Sv1 brainstem
adenosine abundance was un-altered (Fig. 1C) in 129Sv1
mice; while adenosine abundance was decreased in the
brainstem of C57BL/6J mice during the dependent state,
administration protocol and at the same time as the
129Sv1 mice. We previously demonstrated that co-
administration of ondansetron (1 mg/kg IP with morphine
C57BL/6J mice (Chu et al., 2009). However, ondansetron
co-administration with morphine did not alter the decrease
in brainstem adenosine abundance that is induced by
not develop physical
to thesame morphine
Fig. 1. (A) The 4-day protocol for developing morphine dependence, and for inducing natural or naloxone-induced withdrawal is shown. (B)
Adenosine abundance (± SEM) in brainstem tissue obtained from 4 groups of C57BL/6J mice (n = 8 per group). (C) Adenosine abundance
(± SEM) in brainstem tissue obtained from the indicated groups (n = 8 per group) of 8-week-old male C57BL/6J or 129Sv1 mice is shown.
Ondansetron (4 mg/kg IP) was also co-administered with each morphine dose to C57BL/6J mice. The p-value, which is calculated relative to control
mice for each of the groups, is also shown. The data presented in each of panel were produced in independent experiments with different LC/MS
analyses. Due to differences in metabolite extraction efficiency and in detection sensitivity, the measurements obtained in one experiment cannot be
directly compared across different experiments, which are shown in different panels. However, the adenosine abundance in brainstem tissue
obtained from 129Sv1 mice in the basal state was significantly below the level in basal C57BL/6J mice.
M. Wu et al./Neuroscience 228 (2013) 235–242
morphine and naloxone treatment (Fig. 1C). Thus, there is
a specific decrease in brainstem adenosine levels in
C57BL/6J mice during the morphine dependence and
withdrawal states; this metabolomic change specifically
occurs in the opiate-dependent strain. While ondansetron
reduces behavioral aspects of dependence, it acts distal
to the site where opiates alter adenosine abundance.
Adenosine deaminase and adenosine kinase (ADK)
can reduce adenosine by forming inosine and AMP,
respectively. However, ADK is the key regulator of
adenosine metabolism in the adult brain (Boison, 2006).
Consistent with ADK playing an important role in the
tissues during the periods of morphine dependence or
during the two different withdrawal states was not
altered (Fig. 3). To study the role of adenosine and ADK
in opiate responses, we characterized opiate responses
in two mouse strains with altered levels of ADK
expression (Li et al., 2008). In both of these lines, the
expression changes (Studer et al., 2006; Masino et al.,
2011), has been replaced by a constitutively over-
expressed transgene (Li et al., 2007, 2008). Both lines
were maintained on the C57BL/6J background that
exhibits a high degree of opiate dependence. Adk-tg
mice have globally increased ADK expression in brain,
including brainstem; while fb-Adk-def have an identical
level of ADK over-expression of ADK throughout the
entire basal and midbrain regions, but have reduced
ADK expression within the entire dorsal telencephalon
(Li et al., 2008) (Fig. 4A). We first measured the jumping
behavior precipitated by naloxone administration to
opiate dependent control, Adk-tg, and fb-Adk-def mice.
There was a very significant reduction (p < 0.0004) in
the withdrawal response exhibited by Adk-tg and fb-
(Fig. 4B). The similarly reduced withdrawal response in
transgenic mice with increased (Adk-tg) or decreased
expression(p > 0.05)
Fig. 2. Confirmation of the identity of adenosine in extracts using a chemical standard. (A) The extracted ion chromatogram of adenosine in a
brainstem extract (red) overlapped with that of an adenosine standard (black), which indicates that they have identical retention times. (B)
Confirmation of the identity of adenosine by LC/MS/MS analysis. The upper (red) and lower (black) panels show the collision-induced dissociation
(CID) spectrum of adenosine in a brainstem sample and of an adenosine standard, respectively. The collision energy was set at 28 V. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
ControlDependence Naloxone Withdrawal
Fig. 3. Inosine abundance (± SEM) in C57BL/6J brainstem tissue
obtained from the indicated 4 groups of mice (n = 8 per group). The
p-value and fold change, which is calculated relative to that in the
control mice for each of the 3 groups analyzed, are also shown.
238M. Wu et al./Neuroscience 228 (2013) 235–242
indicates that other brain regions affect the withdrawal
Then, adenosine abundance in the brainstem of
control C57BL/6J and fb-Adk-def mice was measured in
the basal state and in morphine-dependent mice after
abundance was decreased in C57BL/6J mice during
naloxone-precipitated withdrawal (p = 0.028) relative to
basal. Although adenosine abundance in brainstem was
reduced in fb-Adk-def mice in the basal state relative to
control C57BL/6J mice, it was not further decreased in
the withdrawal state, which is consistent with the
elimination of the endogenous Adk-gene (Fig. 4C). This
indicates that ADK activity affects the development of
Fig. 4. (A) Representative immunohistochemical images of sagittal brain sections from wildtype (C57BL/6J), fb-Adk-def and Adk-tg mice. The
sections were stained with diamino-benzidine hydrochloride (DAB) for ADK immunoreactivity. (B) C57BL/6J, Adk-tg, and fb-adk-def mice (which
over-express ADK in basal and midbrain regions but have reduced ADK expression in forebrain) were treated for 4 days with morphine to establish
physical dependence (n = 8–9 mice per group). On day 4, the number of jumps made during the 15-min period after naloxone injection was
measured as an indicator of opioid dependence. The data represent the mean number of jumps for each indicated group ± SEM. (C) Adenosine
abundance (± SEM) in the brainstem (B). (D,E) The effect of selective adenosine receptor agents in morphine dependent mice on (D) naloxone-
precipitated withdrawal behavior or (E) withdrawal-induced hyperalgesia. A1R agonist 2-chloro-N6-cyclopentyladenosine (CCPA 0.5 mg/kg i.p.),
A2AR selective antagonist SCH 58261 (1 mg/kg i.p.) or vehicle (saline) was administered 15 min prior to behavior testing. Data represent the
mean ± SEM for n = 8 mice per group; and⁄⁄p < 0.01,⁄⁄⁄p < 0.001.
M. Wu et al./Neuroscience 228 (2013) 235–242
narcotic drug dependence and the level of adenosine in
the brainstem. It is also noteworthy that the basal
adenosine levels in both 129Sv1 and fb-Adk-def mice
exhibited a greater level of narcotic dependence after
four days of opiate administration (Figs. 1C and 4C).
adenosine levels in 129Sv1 and fb-Adk-def mice were
not changed after morphine administration.
It was surprising that the opiate-induced change in the
ondansetron, especially since ondansetron alleviated
opiate dependence and withdrawal symptoms. However,
it was possible that ondansetron could act downstream
of the opiate-induced change in adenosine, possibly
through an effect on adenosine receptors. To investigate
this possibility, we examined the effect that drugs acting
behaviors. Consistent with the results observed by
others (Zarrindast et al., 1999), administration of an
adenosine receptor1(A1) agonist significantly attenuated
administration of an adenosine A2a
antagonist also decreased opioid withdrawal symptoms
opioid withdrawal complication (Fig. 4E). These results
demonstrate that adenosine receptors have a strong
effect on reducing withdrawal behaviors.
was notaltered by
This study demonstrates that semi-targeted metabolomic
neurobehavioral responses. It also provides the first
decreases adenosine abundance in brainstem. The
decrease in narcotic drug withdrawal behavior in mice
adenosine levels or after administration of pharmacologic
agents acting on adenosine receptors demonstrates the
importance of this opiate-induced metabolomic change.
contributes (at least in part) to the neurobehavioral
features of addiction and withdrawal. Adenosine is an
important inhibitory neuromodulator; it inhibits glutamate
release (Brambilla et al., 2005) and the post-synaptic
action of excitatory neurotransmitters via activation of
A1R (Dunwiddie and Masino, 2001), which are densely
expressed in the brainstem (Reppert et al., 1991; Dixon
et al., 1996). Adenosine (Ahlijanian and Takemori, 1985;
Kaplan and Coyle, 1998; Salem and Hope, 1999),
adenosine receptors (Kaplan et al., 1994; Kaplan and
Sears, 1996; Salem and Hope, 1997) and A2afunction
(Latini and Pedata, 2001; Yao et al., 2006; Brown and
Short, 2008; Castane et al., 2008; Brown et al., 2009)
have been previously linked with opiate responses and
addiction. Although the relationship is more complex and
variable, partly because A2aexpression is primarily in the
striatum (Dixon et al., 1996), A2afunction has also been
linked with opiate responses and addiction (Stella et al.,
2003; Yao et al., 2006; Castane et al., 2008; Brown
et al., 2009). The opiate-induced decrease in adenosine
occurs within the brainstem, a region that has been
Bhargava, 1989; Tao et al., 1998). The withdrawal
response from diazepam, ethanol, nicotine or cocaine in
mice was previously shown to be antagonized by
ondansetron injection into the amygdala and dorsal
accumbens and striatum were ineffective (Costall et al.,
The extra-cellular adenosine concentration is highly
dynamic, and can increase in response to hypoxia or
concentration is determined by the rate of inward
adenosine flux, which is mediated by equilabrative
intracellular adenosine levels. The intracellular adenosine
concentration depends largely on metabolic clearance
through ADK, which converts adenosine to AMP. Thus,
the rate of intracellular adenosine metabolism and the
intracellular adenosine concentration is controlled by
ADK activity. By this mechanism, intracellular adenosine
is the primary determinant of extra-cellular adenosine
concentration (Greene, 2011).
Although we do not fully understand the mechanism
(or even the required magnitude) through which a
change in brain adenosine tone mediates opiate-induced
behaviors, three key observations emerge from the
metabolomic data presented here. (i) Morphine induces
a decrease in brainstem adenosine tone in C57BL/6J
mice, which become morphine dependent. (ii) The basal
brainstem adenosine levels in 129Sv1 and fb-Adk-def
mice, which do not become morphine dependent, were
lower than those in C57BL/6 mice. (iii) Brainstem
adenosine levels in 129Sv1 and fb-Adk-def mice did not
change after opiate administration. These observations
can be unified by the concept that drug-induced changes
in adenosine tone affect dependence. In C57BL/6J mice,
which are dependence prone, morphine strongly down-
regulates brain adenosine tone. This could facilitate the
development of dependence via reduced A1 receptor
signaling and enhanced neuronal excitability (Dunwiddie
and Masino, 2001; Greene, 2011). In contrast, little
dependence develops in ADK transgenic or 129/Sv mice
that have a low basal adenosine tone, which is not
altered after morphine administration. Since these mice
do not experience a morphine-induced change in brain
adenosine tone, this could make them less likely to
develop dependence. The decrease in dependence
observed after treatment with an A1R agonist is also
consistent with this concept; agonist-induced adenosine
signaling could also decrease neuronal excitability. The
effect that A2areceptor antagonists have on decreasing
morphine withdrawal reveals that there may be more
complexity in the effect of adenosine on morphine
responses. Conflicting results have been obtained using
other A2a antagonists, and some were reported to
augment opioid dependence (Kaplan and Sears, 1996),
but they lacked specificity for adenosine receptors.
240 M. Wu et al./Neuroscience 228 (2013) 235–242
Morphine withdrawal in rats was also attenuated after
administration of the A2a antagonist used here (Stella
et al., 2003). Of note, Halimi et al. (2000) reported a very
modest (20%) increase in adenosine levels in striatal
tissue after morphine administration, which is where A2a
receptors are expressed; but this region was not
analyzed here. It is possible that there is regional
variation in the effects of adenosine signaling, which
could be due to regional variation in A2aexpression.
Of note, adenosine has an important role in sleep
regulation; increased adenosine levels promote sleep,
while adenosine receptor antagonists have been shown
to induce wakefulness (Porkka-Heiskanen et al., 1997;
Bjorness and Greene, 2009; Palchykova et al., 2010). In
rats, opioids have been shown to disrupt sleep, and to
decrease adenosine levels in brain regions that regulate
sleep (Nelson et al., 2009; Gauthier et al., 2011).
Similarly, opioids disrupt human sleep (Kay et al., 1981),
and major disruptions of sleep occur during human opiate
withdrawal (Oyefeso et al., 1997; Beswick et al., 2003).
Adenosine also plays an important role in the regulation
of nociception (Sawynok, 1998), and opioid withdrawal
response. Seizures are also an important part of narcotic
neonates born to mothers that chronically use narcotic
drugs (O’Grady et al., 2009). Increased adenosine
clearance due to over-expression of transgenic ADK in
mouse brain has also been shown to induce spontaneous
seizure activity (Fedele et al., 2005; Li et al., 2008), which
is dependent upon A1R function (Masino et al., 2011).
Further studies are required to better define the affect of
opiates on adenosine and other metabolites. However,
these results raise the intriguing possibility that opiate-
induced alterations in brain adenosine levels could
contribute to the hyperalgesia, disrupted sleep and
seizures that are characteristic of opiate withdrawal.
Our results demonstrate metabolomic analysis can
provide insight into the mechanisms mediating opiate
responses. Furthermore, our results also indicate that
opiate-induced changes in brain adenosine tone may
mediate clinically important opiate responses. Although
further studies are required, an increased understanding
of this mechanism could produce new strategies for
addressing the public health concern created by narcotic
Acknowledgements—G.P. was partially supported by funding
from a transformative RO1 award (1R01DK090992) provided
by the NIDDK.
phenylisopropyl)-adenosine (PIA) and caffeine on nociception
and morphine-induced analgesia, tolerance and dependence in
mice. Eur J Pharmacol 112:171–179.
Beswick T, Best D, Rees S, Bearn J, Gossop M, Strang J (2003)
Major disruptions of sleep during treatment of the opiate
withdrawal syndrome: differences between methadone and
lofexidine detoxification treatments. Addict Biol 8:49–57.
MK,Takemori AE(1985) Effectsof(?)-N6-(R-
Bjorness TE, Greene RW (2009) Adenosine and sleep. Curr
Boison D (2006) Adenosine kinase, epilepsy and stroke: mechanisms
and therapies. Trends Pharmacol Sci 27:652–658.
Brambilla D, Chapman D, Greene R (2005) Adenosine mediation of
presynaptic feedback inhibition of glutamate release. Neuron
Brown RM, Short JL (2008) Adenosine A(2A) receptors and their role
in drug addiction. J Pharm Pharmacol 60:1409–1430.
Brown RM, Short JL, Cowen MS, Ledent C, Lawrence AJ (2009) A
differential role for the adenosine A2a receptor in opiate
Castane A, Wells L, Soria G, Hourani S, Ledent C, Kitchen I, Opacka-
Juffry J, Maldonado R, Valverde O (2008) Behavioural and
motivational properties are altered in adenosine A(2A) receptor
knockout mice. Br J Pharmacol 155:757–766.
Chu LF, Liang D-Y, Li X, Sahbaie P, D’Arcy N, Liao G, Peltz G, Clark
JD (2009) From mouse to man: the 5-HT3 receptor modulates
physical dependenceon opioid
Costall B, Jones BJ, Kelly ME, Naylor RJ, Onaivi ES, Tyers MB
(1990) Sites of action of ondansetron to inhibit withdrawal from
drugs of abuse. Pharmacol Biochem Behav 36:97–104.
Dixon AK, Gubitz AK, Sirinathsinghji DJ, Richardson PJ, Freeman TC
(1996) Tissue distribution of adenosine receptor mRNAs in the
rat. Br J Pharmacol 118:1461–1468.
Dodrill CL, Helmer DA, Kosten TR (2011) Prescription pain
medication dependence. Am J Psychiatry 168:466–471.
Dunwiddie TV, Masino SA (2001) The role and regulation of
adenosine in the central nervous system. Annu Rev Neurosci
Fedele DE, Gouder N, Guttinger M, Gabernet L, Scheurer L, Rulicke
T, Crestani F, Boison D (2005) Astrogliosis in epilepsy leads to
aggravation. Brain 128:2383–2395.
Gauthier EA, Guzick SE, Brummett CM, Baghdoyan HA, Lydic R
(2011) Buprenorphine disrupts sleep and decreases adenosine
concentrations in sleep-regulating brain regions of Sprague
Dawley rat. Anesthesiology 115:743–753.
Greene RW (2011) Adenosine: front and center in linking nutrition
and metabolism to neuronal activity. J Clin Invest 121:2548–2550.
Gulati A, Bhargava HN (1989) Brain and spinal cord 5-HT2 receptors
of morphine-tolerant-dependent and -abstinent rats. Eur J
Guo K, Li L (2009) Differential 12C-/13C-isotope dansylation labeling
and fast liquid chromatography/mass spectrometry for absolute
and relative quantification of the metabolome. Anal Chem
Halimi G, Devaux C, Clot-Faybesse O, Sampol J, Legof L, Rochat H,
Guieu R (2000) Modulation of adenosine concentration by opioid
receptor agonists in rat striatum. Eur J Pharmacol 398:217–224.
Kaplan GB, Coyle TS (1998) Adenosine kinase inhibitors attenuate
opiate withdrawal via adenosine receptor activation. Eur J
Kaplan GB, Leite-Morris KA, Sears MT (1994) Alterations of
adenosine A1 receptors in morphine dependence. Brain Res
Kaplan GB, Sears MT (1996) Adenosine receptor agonists attenuate
and adenosine receptor antagonists exacerbate opiate withdrawal
signs. Psychopharmacology (Berl) 123:64–70.
Kay DC, Pickworth WB, Neider GL (1981) Morphine-like insomnia
from heroin in nondependent human addicts. Br J Clin Pharmacol
Latini S, Pedata F (2001) Adenosine in the central nervous system:
Li T, Quan Lan J, Fredholm BB, Simon RP, Boison D (2007)
Adenosine dysfunctionin astrogliosis:
generation? Neuron Glia Biol 3:353–366.
kinase,resulting in seizure
M. Wu et al./Neuroscience 228 (2013) 235–242
Li T, Ren G, Lusardi T, Wilz A, Lan JQ, Iwasato T, Itohara S, Simon
RP, Boison D (2008) Adenosine kinase is a target for the
prediction and prevention of epileptogenesis in mice. J Clin Invest
Liang D, Liao G, Wang J, Usuka J, Guo YY, Peltz G, Clark JD (2006a)
A genetic analysis of opioid-induced hyperalgesia in mice.
Liang DY, Li X, Clark JD (2011) 5-Hydroxytryptamine type 3 receptor
modulates opioid-induced hyperalgesia and tolerance in mice.
Liang DY, Liao G, Lighthall G, Peltz G, Clark JD (2006b) Genetic
variants of the p-glycoprotein gene Abcb1b modulate opioid-
induced hyperalgesia, tolerance and dependence. Pharmacogenet
Masino SA, Li T, Theofilas P, Sandau US, Ruskin DN, Fredholm BB,
Geiger JD, Aronica E, Boison D (2011) A ketogenic diet
suppresses seizures in mice through adenosine A receptors. J
Clin Invest 121:2679–2683.
Nelson AM, Battersby AS, Baghdoyan HA, Lydic R (2009) Opioid-
induced decreases in rat brain adenosine levels are reversed by
inhibiting adenosine deaminase. Anesthesiology 111:1327–1333.
O’Grady MJ, Hopewell J, White MJ (2009) Management of neonatal
abstinence syndrome: a national survey and review of practice.
Arch Dis Child Fetal Neonatal Ed 94:F249–252.
Oyefeso A, Sedgwick P, Ghodse H (1997) Subjective sleep–wake
parameters in treatment-seeking opiate addicts. Drug Alcohol
Palchykova S, Winsky-Sommerer R, Shen HY, Boison D, Gerling A,
Tobler I (2010) Manipulation of adenosine kinase affects sleep
regulation in mice. J Neurosci 30:13157–13165.
Poree LR, Guo TZ, Kingery WS, Maze M (1998) The analgesic
potency of dexmedetomidine is enhanced after nerve injury: a
possible role for peripheral alpha2-adrenoceptors. Anesth Analg
Porkka-Heiskanen T, Strecker RE, Thakkar M, Bjorkum AA, Greene
RW, McCarley RW (1997) Adenosine: a mediator of the sleep-
wakefulness. Science 276:
Reppert SM, Weaver DR, Stehle JH, Rivkees SA (1991) Molecular
cloning and characterization of a rat A1-adenosine receptor that is
widely expressed in brain and spinal cord. Mol Endocrinol
Salem A, Hope W (1997) Effect of adenosine receptor agonists and
antagonists on the expression of opiate withdrawal in rats.
Pharmacol Biochem Behav 57:671–679.
Salem A, Hope W (1999) Role of endogenous adenosine in the
expression of opiate withdrawal in rats. Eur J Pharmacol
Sawynok J (1998) Adenosine receptor activation and nociception.
Eur J Pharmacol 347:1–11.
Smith SB, Marker CL, Perry C, Liao G, Sotocinal SG, Austin JS,
Melmed K, David Clark J, Peltz G, Wickman K, Mogil JS (2008)
Quantitative trait locus and computational mapping identifies
Kcnj9 (GIRK3) as a candidate gene affecting analgesia from
multiple drug classes. Pharmacogenet Genomics 18:231–241.
Stella L, De Novellis V, Vitelli MR, Capuano A, Mazzeo F, Berrino L,
Rossi F, Filippelli A (2003) Interactive role of adenosine and
dopamine inthe opiate
Schmiedebergs Arch Pharmacol 368:113–118.
Studer FE, Fedele DE, Marowsky A, Schwerdel C, Wernli K, Vogt K,
Fritschy JM, Boison D (2006) Shift of adenosine kinase
Tao R, Ma Z, Auerbach SB (1998) Alteration in regulation of serotonin
release in rat dorsal raphe nucleus after prolonged exposure to
morphine. J Pharmacol Exp Ther 286:481–488.
Yao L, McFarland K, Fan P, Jiang Z, Ueda T, Diamond I (2006)
Adenosine A2a blockade prevents synergy between mu-opiate
and cannabinoid CB1 receptors and eliminates heroin-seeking
behaviorin addicted rats.
Zarrindast MR, Naghipour B, Roushan-zamir F, Shafaghi B (1999)
Effects of adenosine receptor agents on the expression of
morphine withdrawal in mice. Eur J Pharmacol 369:17–22.
(Accepted 13 October 2012)
(Available online 22 October 2012)
242M. Wu et al./Neuroscience 228 (2013) 235–242