research article electroacupuncture reduces cocaine...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 134610 13 pageshttpdxdoiorg1011552013134610

Research ArticleElectroacupuncture Reduces Cocaine-Induced Seizures andMortality in Mice

Yi-Hung Chen1 Boris Ivanic12 Chieh-Min Chuang1 Dah-Yuu Lu3 and Jaung-Geng Lin4

1 Graduate Institute of Acupuncture Science China Medical University Taichung 40402 Taiwan2 Childrenrsquos University Hospital Limbova 1 Bratislava Slovakia3 Graduate Institute of Neural and Cognitive Sciences China Medical University Taichung 40402 Taiwan4 School of Chinese Medicine China Medical University Taichung 40402 Taiwan

Correspondence should be addressed to Yi-Hung Chen yihungchenmailcmuedutw andJaung-Geng Lin jglinmailcmuedutw

Received 9 January 2013 Accepted 17 February 2013

Academic Editor Gerhard Litscher

Copyright copy 2013 Yi-Hung Chen et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The aims of this study were to characterize the protective profile of electroacupuncture (EA) on cocaine-induced seizures andmortality inmiceMicewere treatedwith EA (2Hz 50Hz and 100Hz) or they underwent needle insertionwithout anesthesia at theDazhui (GV14) and Baihui (GV20) acupoints before cocaine administration EA at 50 Hz applied to GV14 and GV20 significantlyreduced the seizure severity induced by a single dose of cocaine (75mgkg ip) Furthermore needle insertion into GV14 andGV20 and EA at 2 Hz and 50 Hz at both acupoints significantly reduced themortality rate induced by a single lethal dose of cocaine(125mgkg ip) In the sham control group EA at 50Hz applied to bilateral Tianzong (SI11) acupoints had no protective effectsagainst cocaine In addition EA at 50Hz applied toGV14 andGV20 failed to reduce the incidence of seizures andmortality inducedby the local anesthetic procaine In an immunohistochemistry study EA (50Hz) pretreatment at GV14 andGV20 decreased cocaine(75mgkg ip)-induced c-Fos expression in the paraventricular thalamus While the dopamine D

3receptor antagonist SB-277011-

A (30mgkg sc) did not by itself affect cocaine-induced seizure severity it prevented the effects of EA on cocaine-induced seizuresThese results suggest that EA alleviates cocaine-induced seizures and mortality and that the dopamine D

3receptor is involved at

least in part in the anticonvulsant effects of EA in mice

1 Introduction

11 Cocaine-Induced Seizures and Death Cocaine is a widelyabused psychomotor stimulant [1 2] In addition to itsaddiction liability the abuse of cocaine is associated withan array of medical complications [3] Generalized tonic-clonic seizures and status epilepticus are well-documentedneurologic sequelae of cocaine abuse [3 4] Seizures maybe induced by cocaine after an accidental overdose or afterrecreational use of relatively low doses of cocaine [5 6] Theincidence of seizures in cocaine users has been reported tobe 2ndash10 [7] High doses of cocaine have been associatedwith a disturbingly high number of sudden deaths in adultsa recent study documented that 31 of all sudden deathsin Southwest Spain were related to the use of cocaine [8]

Seizures are considered to be amajor determinant of cocaine-related lethality in humans [4] and animals [9] Suddendeath in cocaine abuse may also be attributed to cardiacarrhythmia and intracerebral hemorrhage [10 11] Currentlyno specific treatment modalities offer protection againstcocaine-induced seizures and death in humans

12 Acupuncture Acupuncture has been used in traditionalChinese medicine for over 2500 years [12] The practice hasrecently gained popularity and is becoming an alternativeandor complementary treatment modality for a variety ofdisorders worldwide [13] In 1997 the US National Institutesof Health stated that acupuncture is a useful method fortreating many conditions and has fewer side effects as

2 Evidence-Based Complementary and Alternative Medicine

comparedwith other contemporarymedical treatments suchas surgery or drugs [13] Two different strategies are usedin acupuncture therapy manual acupuncture (MA) andelectroacupuncture (EA) EA is amodified formof traditionalMA The advantage of EA is in its combined therapeuticeffects of transcutaneous electric nerve stimulation (TENS)and MA Most studies use EA because the latter can bestandardized by frequency voltage waveform length and soforth [14] Studies on animals and humans have demonstratedthat acupuncture results in multiple biological responses [1516] The best characterized mechanism is via the endogenousopioid peptides and their receptors [17]

13 Treating Epilepsy with Acupuncture Acupuncture hasbeen used to treat epilepsy in China for thousands of yearsMany reports have suggested that acupuncture TENS andother alternative therapies may produce positive effects inepilepsy [19] although some studies disagree [20]Themajoradvantage of acupuncture is the absence of side effectsIn the clinic acupuncture at specific acupoints such asthe Zusanli (ST36) Dazhui (GV14) and Baihui (GV20)has significantly ameliorated symptoms of epilepsy [21ndash23]Although some evidence supports this contention [24ndash26]the precise mechanism remains unknown It is noted thatacupuncture to GV20 point has been used to treat loss ofconsciousness and tinnitus in addition to relief of mentalabstraction sluggish speech and hysteria [27] Recent studieshave shown that EA inhibits seizures in experimental ratmodels These protective effects may be related to increasedconcentrations of inhibitory amino acids [28] decreasedlevels of nitric oxide in the central nervous system (CNS)[26 29] or increased cellular glutamic acid decarboxylase-67 mRNA expression thereby increasing the production of120574-aminobutyric acid (GABA) [30] The possibility that EAprotects against cocaine-induced seizures and death has notyet been explored

14 Acupuncture and Abused Drugs Acupuncture has beenwidely used throughout Asian countries for treating manyfunctional disorders such as substance abuse and mental ill-ness [31] Some preclinical data have shown that acupuncturecanmodify themorphinewithdrawal syndrome and suppressalcohol drinking behavior in rats [32 33] Further evidencesuggests that acupuncture at a specific acupoint (ShenmenHT7) attenuates the ethanol-induced dopamine release in thenucleus accumbens through the GABAB receptor [34] andsuppresses c-Fos expression in the nucleus accumbens andventromedial striatum following a nicotine challenge in ratssensitized to nicotine [35]

15 c-Fos Expression Expression of c-fos gene or Fos proteinis widely used as a marker to identify neuronal pathwaysinvolved in the integration of noxious inputs [36ndash40] Highdoses of cocaine have been reported to induce c-fos mRNAexpression in many brain regions [41]

In this study EA experimentswere conducted to (1) estab-lish the applicability of EA to cocaine-induced seizure and

mortality in a mouse model (2) determine the frequency-dependent antiseizure activity of EA and (3) correlate theefficacy of EA with the expression of Fos protein in the brain

2 Methods

21 Laboratory Animals Male ICR mice (28ndash35 g BioLascoTaiwan Co Ltd Taiwan) were housed under a 1212 hlightdark cycle with food and water available ad libitum inour animal facility for at least 4 days prior to the experimentswhich were conducted between 1000 and 1700 hThe exper-imental procedures were approved by the China MedicalUniversity Institutional Animal Care and Use Committee inaccordance with the Chinese Taipei Society of LaboratoryAnimal Sciences guidance on care and use of laboratoryanimals Experiments were designed to keep the number ofmice at aminimumand care was taken tominimize suffering

22 Electroacupuncture For the EA treatment animals werecovered by paraffin film and restrained by tapes withoutanesthesia A pair of stainless steel acupuncture needles(Tianjin HaingLimSou Won Medical CO Ltd Gauge 40)were inserted 3-4mm deep into the murine equivalent ofthe human GV14 and GV20 that is (1) the skin at thelocation between the last cervical and the first thoracicvertebral spinous processes at the midline of the back whichis equivalent to the human GV14 and (2) at the vertex ofthe parietal bone that is the midpoint of the connectingline between the auricular apices which is equivalent to thehuman GV20 acupoints [42 43] EA stimuli were deliveredby an EA Trio 300 stimulator (Ito Japan) at 1mA intensitiesfor a 15min duration at a frequency of 2 50 or 100Hz witha pulse width of 150 120583sThe two electrodes were connected tothe needles which were inserted into GV20 and GV14 In thecontrol group animals were also covered by paraffin film andrestrained by tape for 15min [44] A sham EAwas performedby bilateral insertion of a pair of stainless steel acupunctureneedles approximately 3-4mm deep into the middle of eachscapula which is equivalent to the human Tianzong (SI11)

23 Behavioral Study Following the injection of cocaineor saline animals were placed in a plastic observation boxand seizure scores were assessed by the Itzhak scale whichcategorizes five stages according to their severity over a30min period [18]The Itzhak stages of seizure are as follows

Stage 1 Normal behavior (moving about the cage sniffingand rearing)

Stage 2 Hyperactivity (running movement characterized byrapid changes in position)

Stage 3 Animal remains in the same place for several secondswith fast repetitive movements of the head face mouth orforelimb as well as head nodding

Stage 4 Forelimb clonus and rearing

Evidence-Based Complementary and Alternative Medicine 3

Stage 5 Full motor seizures characterized by clonus offorelimbs and hindlimbs flexion of head or entire body andcomplete loss of postural control

24 Immunohistochemistry Procedure The immunohisto-chemistry procedure was similar to that described by Inanet al [45] Two hours after the injection of saline or cocaineanimalswere deeply anesthetizedwith urethane (12 gkg ip)and perfused intracardially with ice-cold 01M phosphate-buffered saline (PBS) followed by 4 paraformaldehydein 01M PBS The brains were removed postfixed for 2 hand kept in 30 sucrose solution overnight Brain sectionsof 50120583m thickness were cut with a cryostat (LEICA CM3050) Free-floating sections were stored in PBS at 4∘C untilimmunohistochemistry was performedThree brain sectionsfrom minus2255mm to minus1856mm caudal to bregma wererandomly selected for immunohistochemistry proceduresTissues were processed for c-Fos immunoreactivity by theavidin-biotin complex procedure [46] Tissues were initiallytreated with 3 H

2O2to reduce endogenous peroxidase

activity then washed twice for 10min with PBS and blockedwith 20 normal goat serum (1 20) for 2 h at room tem-perature The sections were then incubated on a shaker for2 d at 4∘C with a rabbit c-Fos antibody (1 1000 dilution) (sc-52 Santa Cruz USA) After thorough rinsing sections wereincubated in biotinylated anti-rabbit immunoglobulin G sec-ondary antibody (1 300 dilution Vector Laboratories USA)for 2 h at room temperature Following two 10min PBS rinsesthe sections were incubated in a complex of avidin-biotin-peroxidase in 1 300 solution at room temperature for 90min(Vectastain ABC Elite kit Vector Laboratories) Followingthree 10min washes in Tris-buffered saline the sections wereplaced in a 005 diaminobenzidine (Sigma)0001 H

2O2

solution for 4-5min and washed again with Tris-bufferedsaline 3 times for 10min each Sections were mounted onslides with 025 gel alcohol air-dried and dehydratedwith graded alcohol (50 70 95 and 100 for 6mineach) followed by xylene (3 times for 10min each) and thecoverslip was placed using Permount c-Fos positive nucleiwere observed under a light microscope and counted at 200xmagnification c-Fos immunoreactive nuclei were counted onthe captured images using MetaMorph (Universal ImagingCorp) software Bilateral calculations were performed onthe paraventricular thalamus amygdala area and caudoputa-menThe number of immunoreactive nuclei was averaged foreach mouse

25 Drugs and Chemicals Cocaine-HCl was purchased fromthe National Bureau of Controlled Drugs Department ofHealth Taipei Taiwan Freshly dissolved in 09 NaCl alltest agents were administered in a dosevolume of 10mgmLCocaine at the dose of 75mgkg causes clonic seizures inmice [48] Cocaine at 125mgkg is considered to be a lethaldose [49] In this study cocaine at 75mgkg and 125mgkgwas injected intraperitoneally to induce seizures and deathProcaine HCl was purchased from Sigma Chemical Co (StLouis MO USA) Procaine (250mgkg and 400mgkg ip)was used to induce seizures and death in mice respectively

[50] SB-277011-A (30mgkg sc) was purchased from TocrisBioscience (Ellisville MO USA) and was used to block thedopamine D

3receptor [51]

26 Statistical Analysis Data were expressed as means andthe standard error of the mean (SEM) Group comparisonsfor seizure scores and the number of cocaine-induced c-Fospositive nuclei were evaluated by one-way ANOVA followedby Tukeyrsquos post-hoc test Group comparisons for mortalityrates and seizure incidence were evaluated by chi-square testor Fisherrsquos exact test Significance was considered at 119875 lt 005for all tests Statistical analysis was performed by SPSS version180 (SPSS Inc Chicago IL USA)

3 Results

31 Positioning of Acupuncture Needles We used X-rayimages to ensure accurate positioning of acupuncture nee-dles As shown in Figure 1(a) a mouse was anesthetized withurethane and two acupuncture needles were inserted into theGV14 and GV20 acupoints Figure 1(b) shows the positionof the acupuncture needles as revealed by X-ray Figure 1(c)shows the positions of GV14 GV20 and SI11 in mice

32 Effects of EA on Cocaine-Induced Seizures Firstly theeffects of EA were examined on seizures induced by a singledose of cocaine (75mgkg) in six groups of mice (a controlgroup three EA treatment groups a sham EA group and aneedle insertion group) In the control group animals wererestrained with the same procedure as that used with theEA group but without EA In the EA treatment groups EA(2Hz 50Hz and 100Hz) was applied for 15min to GV14 andGV20 acupoints prior to cocaine injection In the sham EAgroup mice received EA (50Hz) applied to bilateral SI11 Inthe needle insertion group needles were inserted into GV14andGV20 acupoints but without electrical stimulation Afterundergoing control EA sham or needle insertion treatmentall animals received an intraperitoneal injection of cocaine(75mgkg) Seizure severity was measured by the 5-stagecocaine seizure scale published by Itzhak [18]

As shown in Figure 2 the average seizure score wassignificantly lower in animals treated with EA (50Hz) com-pared with those in the control and sham EA groups Nosuch effects were seen with EA 2Hz EA 100Hz or needleinsertion It appears that EA 50Hz at the GV14 and GV20acupoints significantly reduced seizure scores induced by asingle cocaine administration (75mgkg) whereas EA at 2Hzand 100Hz had no such effect Furthermore cocaine-inducedseizures were not significantly affected by either 15min ofneedle insertion into GV14 and GV20 or by EA at SI11

Table 1 depicts the incidence of seizures (higher or equalto stage 3) induced by cocaine (75mgkg ip) Pretreatmentof animals with EA (50Hz) significantly reduced seizureincidence

33 Effects of EA on Cocaine-Induced Death The effects ofEA on death induced by single administrations of a highdose of cocaine (125mgkg) were investigated in a series


(a) (b)

∙ GV20

∙ GV14

SI11∙

SI11∙

(c)

Figure 1 Photograph (a) and X-ray (b) of a mouse under anesthesia with acupuncture needles inserted into GV14 and GV20 acupoints (c)Mouse schematic showing the location of the acupoints used in the study

5

4

3

2

1

0

lowastlowast lowast

Cocaine 75mgkg

Seiz

ure s

core

s

Cocaine Cocaine Cocaine Cocaine Cocaine Cocaine+ + + + +

119899 = 21

119899 = 20 119899 = 20 119899 = 23 119899 = 20

needleinsertion

EA2 Hz

EA50 Hz

EA100 Hz

shamEA

119899 = 20

Figure 2 Effects of EA at different frequencies (2Hz 50Hz and100Hz) needle insertion exerted at GV14 and GV20 acupoints andsham EA (50Hz at bilateral SI11) on cocaine-induced seizures Micewere administered with cocaine (75mgkg ip) at 1min after EA orneedle insertion Seizure severity was measured by the Itzhak five-stage scale [18] Between-group comparisons for each group wereperformed by one-way ANOVA followed by Tukeyrsquos test (lowast119875 lt 005lowastlowast119875 lt 001 n the number of animals)

of six groups that is control group three EA treatmentgroups a sham EA group and a needle insertion group Afterundergoing treatment all animals received an intraperitonealinjection of cocaine (125mgkg)

As shown in Figure 3 mortality rates were significantlylower in the needle insertion and EA (2 50Hz) groups ascompared to mortality in the control group The EA (2Hz)group mortality rate was also lower than that of the sham EAgroup Needle insertion into GV14 and GV20 as well as EA

lowast lowast

lowastlowastlowast

lowastlowast

80

60

40

20

0

Cocaine 125 mgkgM

orta

lity

()


119899 = 34

119899 = 18 119899 = 18 119899 = 32 119899 = 18 119899 = 17

needleinsertion

EA2 Hz

EA50 Hz

EA100 Hz

shamEA

Figure 3 Effects of EA at different frequencies (2Hz 50Hz and100Hz) needle insertion exerted at GV14 and GV20 acupoints andsham EA (at bilateral SI11 50Hz) on cocaine-induced mortalityrates Mice were administered with cocaine (125mgkg ip) at 1minafter EA or needle insertion andmortality was monitored for 1 hourBetween-group comparisons for each group were performed by chi-square test (lowast119875 lt 005 lowastlowast119875 lt 001lowastlowastlowast119875 lt 0001 n the number ofanimals)

at 2Hz and 50Hz applied to GV14 and GV20 significantlyreduced themortality rate induced by a single dose of cocaine(125mgkg) However EA at 100Hz had no such effect EA atSI11 as a sham control reduced the mortality rate induced bycocaine but did not reach a statistically significant level

34 Effects of EA on Procaine-Induced Seizure and DeathIn addition to inhibiting dopamine uptake cocaine acts as

Evidence-Based Complementary and Alternative Medicine 5M

orta

lity

()

100

80

60

40

20

0Procaine Procaine Procaine

+ +

EA 2 Hz EA 50 Hz119899 = 20

119899 = 21 119899 = 20

Procaine 400 mgkg

Figure 4 Effects of EA at 2Hz and 50Hz exerted at GV14 andGV20 acupoints on procaine-induced (400mgkg ip) mortalityrates At 1min after EA procaine was injected and the mortalitywas monitored for 1 hour Between-group comparisons for eachgroup were performed by chi-square test or Fisherrsquos exact test Nosignificant between-group differences were observed (n the numberof animals)

Table 1 EA (50Hz) applied to GV14 and GV20 reduced theincidence of cocaine-induced seizures but did not alter the incidenceof procaine-induced seizures

Control EA 50HzCocaine (75mgkg ip) 2021 1421lowast

Procaine (250mgkg ip) 89 89The proportion of mice exhibiting seizures is shown here The effects ofcocaine alone and in combination with EA were compared by Fisherrsquos exacttest (lowastP lt 005) Similarly the effects of procaine alone and in combinationwith EA were compared

a local anesthetic by blocking voltage-dependent Na+ chan-nels [52 53] Local anesthetics such as procaine can causeCNS and cardiovascular toxicity when plasma concentrationsare increased by accidental intravenous injection of a lethaldose of procaine [54] However procaine has no CNSstimulant effects compared with those of cocaine Thereforethe effects of EAonprocaine-induced seizures anddeathwereexamined

The seizure incidence of procaine-treated mice (250mgkg ip) is shown in Table 1 When animals were pretreatedwith EA (50Hz) at acupoints GV14 and GV20 the incidencewas not affected Furthermore as shown in Figure 4 mortal-ity rates did not differ significantly between the control groupand EA (2 and 50Hz) groups It appears that EA at 2Hz and50Hz when applied to GV14 and GV20 fails to affect themortality rate associated with procaine (400mgkg ip) inmice

35 EA Attenuation of Cocaine-Induced c-Fos ExpressionThis series of studies was undertaken to test the effects of

Figure 5 Schematic representation of the brain regions inwhich thec-Fos immunoreactive neurons were counted The paraventricularthalamus (in red) the caudoputamen (blue) and the amygdalaarea (including lateral amygdalar nucleus basolateral amygdalarnucleus and basomedial amygdalar nucleus green) are indicatedThe drawing has been modified from the Allen Brain Atlas onlinedatabase (httpwwwbrain-maporg [47])

EA on cocaine-induced (75mgkg) c-Fos expression in themouse brain Mice were divided into 4 groups saline controlEA alone cocaine and cocaine plus EA

All mice in the saline control group and cocaine groupwere restrainedwithout EAMice in the EA alone and cocaineplus EA groups received EA (50Hz) at GV14 and GV20acupoints Following the restraining procedure or EA themice were allowed to recover for 1min before being injectedwith saline (saline control and EA-alone groups) or cocaine(cocaine group and cocaine plus EA groups)

Figure 5 shows the brain regions in which the c-Fosimmunoreactive neurons were counted Results are shownin Figures 6 7 and 8 c-Fos expression was examined inthe three brain areas including paraventricular thalamus(Figure 6) amygdala area (Figure 7) and caudoputamen(Figure 8) Few c-Fos positive nuclei were found in these areasin the saline control group In the EA-alone group c-Fos pos-itive nuclei were found only in the paraventricular thalamus(Figure 6) It appears that EA 50Hz alone had no significanteffect on c-Fos expression except in the paraventricularthalamus In the cocaine group c-Fos positive nuclei werefound in all three areas and at significantly higher levels thanthose seen in the saline control group In the cocaine plus EAgroup although c-Fos positive nuclei were noted in all threebrain areas c-Fos-positive nuclei were significantly decreasedin the paraventricular thalamus (Figure 6) as compared tothose in the cocaine group It appears that pretreatment withEA 50Hz significantly reduced the number of c-Fos positivecells induced by cocaine in the paraventricular thalamusbut not in the amygdala area (Figure 7) and caudoputamen(Figure 8)


Saline EA + saline Cocaine EA + cocaine

(a)

Saline Saline + EA Cocaine Cocaine + EA119899 = 5 119899 = 5 119899 = 14119899 = 9

0

10

20

30

40

50

Num

ber o

f c-F

os p

ositi

ve n

ucle

i

Paraventricular thalamuslowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowast

(b)

Figure 6 (a) Representative photomicrographs of c-Fos expression induced by cocaine in the paraventricular thalamus and the effects ofEA c-Fos expression was not observed among saline controls whereas c-Fos expression was evident in the paraventricular thalamus amonganimals in the EA-alone cocaine-alone (75mgkg ip) and cocaine plus EA groups (b) Effects of EA on controls and cocaine-induced c-Fosexpression in the paraventricular thalamus The numbers of c-Fos positive nuclei were counted and averaged from three randomly chosensections from each animal in each group EA at 50Hz was applied to the GV14 and GV20 acupoints EA increased c-Fos expression in theparaventricular thalamus while EA decreased the number of c-Fos positive nuclei induced by cocaine Between-group comparisons for eachgroup were performed by ANOVA followed by Tukeyrsquos test (lowast119875 lt 005 lowastlowastlowast119875 lt 0001 n the number of animals)

36 EA Effects on Cocaine-Induced Seizures Blocked by theDopamine D

3Receptor Antagonist SB-277011-A Cocaine

enhances monoamine system activity through the blockadeof dopamine and serotonin reuptake However the mecha-nism of seizures induced by cocaine is complex and involvesinteraction of the drug with several neurotransmitter systemsas well as with voltage-dependent sodium channels [55]It is believed that dopamine receptors play an importantrole in cocaine-induced seizures and death It is known thatthe paraventricular thalamus expresses dopamine D

3mRNA

[56] and we found that EA induced c-Fos expression in theparaventricular thalamus We therefore tested the possibilitythat dopamine D

3receptors maymediate the effects of EA on

cocaine-induced seizuresFour groups were included a control group an SB-

277011-A group an EA group and an SB-277011-A plus EAgroup In the SB-277011-A group and EA group plus SB-277011-A the compound SB-277011-A (30mgkg)was admin-istered subcutaneously for 30min prior to the restrainingprocedure or EA treatment After the restraining procedure


Saline CocaineEA + saline EA + cocaine

(a)

60

50

40

30

20

10

0

lowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowast

Amygdala

Num

ber o

f c-F

os p

ositi

ve n

ucle

i


(b)

Figure 7 (a) Representative photomicrographs of c-Fos expression induced by cocaine in the amygdala and the effects of EAWhereas c-Fosexpression was absent among animals in the saline control and EA plus saline groups c-Fos expression in the amygdala was observed inanimals treated with cocaine (75mgkg ip) and cocaine plus EA (b) Effects of EA on controls and cocaine-induced c-Fos expression in theamygdala The numbers of c-Fos positive nuclei were counted and averaged from three randomly chosen sections from each animal in eachgroup EA at 50Hz was applied to the GV14 and GV20 acupoints Cocaine administration induced a marked level of c-Fos expression whileEA did not significantly decrease the number of c-Fos positive nuclei induced by cocaine Between-group comparisons for each group wereperformed by ANOVA followed by Tukeyrsquos test (lowastlowastlowast119875 lt 0001 n the number of animals)

or EA treatment all animals received an intraperitoneal injec-tion of cocaine (75mgkg) Seizure severity was measured bythe Itzhak five-stage cocaine seizure scale [18]

As shown in Figure 9 the EA (50Hz) group had signif-icantly lower average seizure scores compared with those ofthe remaining three groups Notably seizure scores in the SB-277011-A group and SB-277011-A plus EA group did not differsignificantly from the control group but were significantlyhigher than those in the EA group It appears that while thedopamine D

3receptor antagonist SB-277011-A (30mgkg

sc) did not affect cocaine-induced seizure severity it didprevent the effects of EA on cocaine-induced seizures

4 Discussion

41 EA Decreases Cocaine-Induced Effects The psychostim-ulant and euphoric effects of cocaine are considered to beassociated with the blockade of dopamine uptake in theCNS Acupuncture has been widely used throughout Asiancountries for treating various functional disorders includingsubstance abuse [31] Immunohistochemical investigationsby Lee et al (2009) [57] reported that acupuncture canreduce repeated cocaine-induced locomotor activity in ratsand the expression of tyrosine hydroxylase (TH) in the ratbrain which suggests that acupuncturemay effectively inhibit



(a)


0

10

20

30

40

50

60

Num

ber o

f c-F

os p

ositi

ve n

ucle

i

lowastlowastlowast


lowastlowastlowast

Caudoputamen

(b)

Figure 8 (a) Representative photomicrographs of c-Fos expression induced by cocaine in the caudoputamen and the effects of EA Whereasc-Fos expressionwas not observed in the saline control and EAplus saline groups c-Fos expression in the caudoputamenwas observed amonganimals treated with cocaine (75mgkg ip) and cocaine plus EA (b) Effects of EA on controls and cocaine-induced c-Fos expression in thecaudoputamen The numbers of c-Fos positive nuclei were counted and averaged from three randomly chosen sections from each animal ineach group EA at 50Hz was applied to the GV14 and GV20 acupoints Cocaine administration induced a marked level of c-Fos expressionwhile EA did not significantly decrease the number of c-Fos positive nuclei induced by cocaine Between-group comparisons for each groupwere performed by ANOVA followed by Tukeyrsquos test (lowastlowastlowast119875 lt 0001 119899 the number of animals)

the behavioral effects of cocaine by modulating the centraldopaminergic system

As mentioned earlier cocaine abuse is associated witha risk of various medical complications including seizuresand death [3] It has previously been reported that cocaineat doses ranging from 25mgkg to 20mgkg (ip) causesconditioned place preference representing the rewardingeffects of cocaine [58] At a dose as high as 40mgkg(ip) cocaine caused seizures in mice [59] The calculatedED50value for cocaine-induced seizures is 5884mgkg [59]

Cocaine at 75mgkg (ip) produces seizures in more than90 of mice [48] Cocaine at 125mgkg is considered to bea lethal dose [49] In the present study we found that EAat 50Hz exerted at GV14 and GV20 acupoints significantly

reduced the seizure severity induced by a single cocaine(75mgkg ip) administration Moreover needle insertioninto GV14 and GV20 as well as EA at 2Hz and 50Hzexerted at GV14 and GV20 acupoints significantly reducedthe mortality rate induced by a single cocaine (125mgkg)administration Conversely EA at 50Hz applied to bilateralSI11 acupoints had no such effects In addition EA failedto protect against procaine-induced seizure incidence andlethality in mice We report here for the first time that EAreduced seizures and mortality induced by a high dose ofcocaine

42 Cocaine-Induced c-Fos Expression and Effects of EAExpression of the c-fos gene or Fos protein is commonly


119899 = 11

119899 = 10

119899 = 12

Cocaine (75 mgkg)

119899 = 9

6

5

4

3

2

1

0

Seiz

ure s

core

s

lowast lowast

lowast

CocaineCocaine+

EA

Cocaine+

EA+

SB-277011-A

Cocaine+

SB-277011-A

Figure 9 Effects of the dopamine D3

receptor antagonistSB-277011-A on the anticonvulsant effects of EA SB-277011-A(30mgkg) was administered subcutaneously 30min prior to thecontrol restraining procedure or EA treatment EA (50Hz) wasapplied for 15min to the GV14 and GV20 acupoints prior to cocaineinjection After the restraining procedure or EA treatment allanimals received an intraperitoneal injection of cocaine (75mgkg)Seizure severity was measured by the Itzhak five-stage scale [18]Between-group comparisons for each groupwere performed by one-way ANOVA followed by Tukeyrsquos test (lowast119875 lt 005 n the number ofanimals)

used as a marker for neuronal activation seizure pathwaysand sites of action of neuroactive drugs [60ndash64] It hasbeen reported that an acute injection of cocaine (65mgkg)in rats increased expression of c-fos mRNA in the dentategyrus of the hippocampus and olfactory bulb and limbiccortical regions as well as the striatum and ventromedialhypothalamic nucleus [41]

In this study we focused on three brain areas the par-aventricular thalamus amygdala and caudoputamen Wefound that cocaine at a dose of 75mgkg (ip) inducedmarked c-Fos expression in all areas Pretreatmentwith 50HzEA significantly reduced the number of c-Fos positive cellsinduced by cocaine in the paraventricular thalamus but notin the amygdala or caudoputamen

The thalamus is considered to be an important interfacebetween the ventral pallidum and the dorsal medial pre-frontal cortex whichmay therefore contribute to the develop-ment of compulsive drug-seeking behavior [65] One thala-mic nucleus that is of particular interest is the paraventricularthalamus a component of the dorsal midline thalamic group[66] The thalamic paraventricular nucleus projects to thenucleus accumbens and other limbic sites including the pre-frontal cortex amygdala and hippocampus these projectionsare predominantly glutamatergic [67] The paraventricularthalamus also receives a dopaminergic innervation in partderived from the ventral tegmental area and paraventricularthalamus neurons expressing dopamine D

3mRNA [56] The

paraventricular thalamus has been implicated in stress reac-tivity [68] reward-seeking behavior [69] and general arousalactivity [70] Furthermore inactivation of the paraventricularthalamus prevents context-induced reinstatement of alcohol-seeking [71] and cocaine-primed reinstatement in rats [72]Paraventricular thalamus lesions block cocaine sensitization[67]

The paraventricular thalamus is related to seizures activ-ity Mraovitch and Calando [73] used immunocytochemistryto determine the regional and temporal distribution of Fosprotein expression in awake and unrestrained rats after a uni-lateral stereotaxic microinjection of the cholinergic agonistcarbachol in the thalamic ventroposterolateral and reticularnuclei previously shown to cause limbic and generalizedseizures [73] They found that paraventricular thalamus acti-vation occurred after 15min after administration of epilepticagents

The amygdala is involved in both temporal lobe epilepsyand in cocaine mechanisms in the brain In particular thecentral nucleus of the amygdala is a highly epileptogenic brainarea and of the amygdaloid nuclei responds most rapidlyto a kindling stimulus [74] The caudoputamen is a part ofthe striatum area and plays an important role in control ofmovement in animals [75ndash77]

As shown by our results and the published literature[41] acute cocaine administration increases c-Fos expressionin many brain areas Among these areas the paraventric-ular thalamus and amygdala are structures associated withseizures while the caudoputamen is associated with move-ment controlWe therefore analyzed c-Fos expression in thesethree specific regions Our results implicate involvement ofthe paraventricular thalamus in the effects of EA on cocaine-induced seizures

43 EA Induces c-Fos Expression in the ParaventricularThala-mus and Dopamine D

3Receptors Are Involved in the Effects

of EA Medeiros et al (2003) reported higher levels of c-Fos expression induced by EA at the ST36 point in animalsthat is in the dorsal raphe nucleus locus coeruleus posteriorhypothalamus and central medial nucleus of the thalamus[78] In the present study we found that EA alone at GV14and GV20 acupoints induced significant c-Fos expression inthe paraventricular thalamus

Recent research indicates that dopamine D3receptors

may play an important role in cocaine-induced seizures[51] The D

3antagonist SB-277011-A has been used to block

dopamine D3receptors at doses ranging from 3mgkg to

30mgkg (sc) [18 79] SB-277011-A prevented the anticon-vulsant effects of the D

3D2receptor agonist (+)-PD-128907

on cocaine-induced seizures Notably the protection affordedby (+)-PD-128907 was eliminated in D

3receptor-deficient

mice whereas the anticonvulsant effects of (+)-PD-128907were preserved in D

2receptor knockout mice

As mentioned above the paraventricular thalamus ex-presses dopamine D

3mRNA while EA alone induces c-

Fos expression in the paraventricular thalamus We thereforesought to determinewhether dopamineD

3receptorsmediate

the effects of EA on cocaine-induced seizures Our results


revealed that while the D3receptor antagonist SB-277011-A

did not affect cocaine-induced seizure severity it preventedthe effects of EA on cocaine-induced seizures This findingsuggests that the D

3receptor is involved at least in part in

the anticonvulsant effects of EAInteresting topics that remain for future studies include

an investigation into whether intraparaventricular thalamusinjection of a D

3receptor antagonist prevents EA-induced

anticonvulsive effects and whether a D3receptor antagonist

antagonizes EA-induced changes to c-Fos expression inthe paraventricular thalamus or has any effect on cocaine-induced mortality

44 A ComplexMechanismUnderlies Cocaine-Induced DeathIt is noted that EA at 50Hz only when exerted at GV14and GV20 acupoints significantly reduced seizure severityinduced by a single cocaine (75mgkg ip) administrationHowever needle insertion into GV14 and GV20 acupoints aswell as EA at 2Hz and 50Hz effectively reduced themortalityrate induced by a single cocaine (125mgkg) administrationAs mentioned earlier seizures are considered to be a majordeterminant of cocaine-related lethality in both humans[4] and animals [9] However the mechanism of suddendeath in cocaine abuse also includes cardiac arrhythmia andintracerebral hemorrhage [10 11] Cocaine produces eupho-ria elation mood elevation alertness attention focusingand fatigue reduction through interactions with monoaminetransporters [80] In addition to the central effects cocainealso blocks norepinephrine uptake and increases sympatheticactivity in the periphery [81] Overstimulation of sympatheticactivity may cause cardiac arrhythmia and intracerebralhemorrhage and hence may contribute to cocaine-inducedfatality [10 11] EA frequency-specific differences observedin protection against seizures and death may be because themechanism that induces death is more complicated thanthat involved in seizures It is recognized that acupuncturecan account for different effects in the autonomic nervoussystem [82 83] For example the GV14 acupoint is capableof increasing parasympathetic activities while simultaneouslysuppressing sympathetic activities [82]Thismay explain whyEA at 2Hz is particularly effective against cocaine-induceddeath

45 EA Frequency The best-known mechanism of EA isvia the endogenous opiates and their receptors Differentkinds of endogenous opioid peptides such as 120573-endorphinenkephalin endomorphin and dynorphin reportedly actin a frequency-dependent manner in EA At a low fre-quency (2Hz) EA accelerated the release of120573-endorphin andenkephalin in the CNS whereas high-frequency EA (100Hz)accelerated the release of dynorphin [17 84ndash86] In thepresent study we found that EA applied to GV14 andGV20 acupoints at the frequency of 50Hz is most effectivein reducing seizure severity induced by a single cocaineadministration There were few reports in the literature as tothe effects of EA 50Hz as compared to those of EA 2 and100Hz However it has been recently reported that EA 60Hzincreases the pain threshold by a greater extent than any

other frequency [87] EA at a frequency of 60Hz induced thesimultaneous release of met-enkephalin 120573-endorphin anddynorphin-A in extensive analgesia-related nuclei and areasof theCNS such as the periaqueductal gray the paraventricu-lar nucleus of the hypothalamus the ventromedial nucleus ofthe hypothalamus the dorsal raphe nucleus and the nucleusraphe magnus amongst others These results suggest thatEA at 60Hz may contribute to optimal analgesic effects Theeffects of EA at 50 or 60Hz deserve to be studied further [87]

No effective treatment currently exists for cocaine-in-duced seizures and death Our results suggest that EA reducesseizure severity and death caused by cocaine in an animalmodel of cocaine abuse We found evidence for involvementof the dopamine D

3receptor at least in part in the anticon-

vulsant effects of EA

Acknowledgments

The authors are grateful to Ms Iona MacDonald and Ms Ya-TingWu for their help in producing the final paperThis workwas supported by Grants NSC 100-2320-B-039-018- and NSC101-2320-B-039-035-MY3 from theNational Science CouncilTaipei Taiwan

References

[1] C Haasen M Prinzleve H Zurhold et al ldquoCocaine use inEuropemdasha multi-centre study methodology and prevalenceestimatesrdquo European Addiction Research vol 10 no 4 pp 139ndash146 2004

[2] R A Lange and L D Hillis ldquoSudden death in cocaine abusersrdquoEuropean Heart Journal vol 31 no 3 pp 271ndash273 2010

[3] N L Benowitz ldquoClinical pharmacology and toxicology of coca-inerdquo Pharmacology and Toxicology vol 72 no 1 pp 3ndash12 1993

[4] W H Spivey and B Euerle ldquoNeurologic complications ofcocaine abuserdquo Annals of Emergency Medicine vol 19 no 12pp 1422ndash1428 1990

[5] A K Dhuna A Pascual-Leone F Langendorf and D CAnderson ldquoEpileptogenic properties of cocaine in humansrdquoNeuroToxicology vol 12 no 3 pp 621ndash626 1991

[6] L D Kramer G E Locke A Ogunyemi and L Nelson ldquoCoca-ine-related seizures in adultsrdquoTheAmerican Journal ofDrug andAlcohol Abuse vol 16 no 3-4 pp 309ndash317 1990

[7] A Pascual-Leone A Dhuna I Altafullah and D C AndersonldquoCocaine-induced seizuresrdquoNeurology vol 40 no 3 part 1 pp404ndash407 1990

[8] J Lucena M Blanco C Jurado et al ldquoCocaine-related suddendeath a prospective investigation in South-West Spainrdquo Euro-pean Heart Journal vol 31 no 3 pp 318ndash329 2010

[9] J D Catravas and IWWater ldquoAcute cocaine intoxication in theconscious dog studies on themechanism of lethalityrdquo Journal ofPharmacology and ExperimentalTherapeutics vol 217 no 2 pp350ndash356 1981

[10] E SecemskyD LangeDDWatersN FGoldschlager andP YHsue ldquoHemodynamic and arrhythmogenic effects of cocaine inhypertensive individualsrdquo Journal of Clinical Hypertension vol13 no 10 pp 744ndash749 2011

[11] S Toossi C PHess N KHills and S A Josephson ldquoNeurovas-cular complications of cocaine use at a tertiary stroke centerrdquo


Journal of Stroke and Cerebrovascular Diseases vol 19 no 4 pp273ndash278 2010

[12] J NWu ldquoA short history of acupuncturerdquo Journal of Alternativeand Complementary Medicine vol 2 no 1 pp 19ndash21 1996

[13] NIH ldquoNIH consensus conference AcupuncturerdquoThe Journal ofthe AmericanMedical Association vol 280 no 17 pp 1518ndash15241998

[14] J G Lin and W L Chen ldquoAcupuncture analgesia a review ofits mechanisms of actionsrdquo The American Journal of ChineseMedicine vol 36 no 4 pp 635ndash645 2008

[15] J H Liu J Yan S X Yi X R Chang Y P Lin and JM Hu ldquoEffects of electroacupuncture on gastric myoelectricactivity and substance P in the dorsal vagal complex of ratsrdquoNeuroscience Letters vol 356 no 2 pp 99ndash102 2004

[16] H R Middlekauff J B Shah J L Yu and K Hui ldquoAcupunctureeffects on autonomic responses to cold pressor and handgripexercise in healthy humansrdquo Clinical Autonomic Research vol14 no 2 pp 113ndash118 2004

[17] J S Han ldquoAcupuncture neuropeptide release produced byelectrical stimulation of different frequenciesrdquo Trends in Neu-rosciences vol 26 no 1 pp 17ndash22 2003

[18] Y Itzhak ldquoAttenuation of cocaine kindling by 7-nitroindazolean inhibitor of brain nitric oxide synthaserdquoNeuropharmacologyvol 35 no 8 pp 1065ndash1073 1996

[19] K J Yin X H Chen R H Wang Z Q Lei and Y W GaoldquoEffect and mechanisms of anti-epilepsy effect by magent-acupuncturerdquo Chinese Acupuncture and Moxibustion vol 10pp 620ndash625 1999

[20] R Kloster P G Larsson R Lossius et al ldquoThe effect ofacupuncture in chronic intractable epilepsyrdquo Seizure vol 8 no3 pp 170ndash174 1999

[21] J L Zhang S P Zhang and H Q Zhang ldquoAntiepileptic effectof electroacupuncture versus vagus nerve stimulation in the ratthalamusrdquoNeuroscience Letters vol 441 no 2 pp 183ndash187 2008

[22] Y S Peng ldquoClinical study on treatment of infantile primaryepilepsy with acupoint catgut-implantation therapyrdquo WorldJournal of Acupuncture-Moxibustion vol 13 no 1 pp 38ndash412003

[23] Y Ren ldquoAcupuncture treatment of Jacksonian epilepsymdashareport of 98 casesrdquo Journal of Traditional Chinese Medicine vol26 no 3 pp 177ndash178 2006

[24] D M Chao G Chen and J S Cheng ldquoMelatonin mightbe one possible medium of electroacupuncture anti-seizuresrdquoAcupuncture and Electro-Therapeutics Research vol 26 no 1-2pp 39ndash48 2001

[25] J G Dos Santos A Tabosa F H M do Monte M M BlancoA de Oliveira Freire and L E Mello ldquoElectroacupunctureprevents cognitive deficits in pilocarpine-epileptic ratsrdquo Neuro-science Letters vol 384 no 3 pp 234ndash238 2005

[26] R Yang Z N Huang and J S Cheng ldquoAnticonvulsion effectof acupuncture might be related to the decrease of neuronaland inducible nitric oxide synthasesrdquo Acupuncture and Electro-Therapeutics Research vol 25 no 3-4 pp 137ndash143 2000

[27] Y H Chang M T Hsieh and J T Cheng ldquoIncrease oflocomotor activity by acupuncture on Bai-Hui point in ratsrdquoNeuroscience Letters vol 211 no 2 pp 121ndash124 1996

[28] Q Li J C Guo H B Jin J S Cheng and R Yang ldquoInvolvementof taurine in penicillin-induced epilepsy and anti-convulsion ofacupuncture a preliminary reportrdquo Acupuncture and Electro-Therapeutics Research vol 30 no 1-2 pp 1ndash14 2005

[29] Z N Huang R Yang G Chen and J S Cheng ldquoEffect ofelectroacupuncture and 7-NI on penicillin-induced epilepsyand their relation with intrahippocampal no changesrdquo ActaPhysiologica Sinica vol 51 no 5 pp 508ndash514 1999

[30] J Guo J Liu W Fu et al ldquoThe effect of electroacupunctureon spontaneous recurrent seizure and expression of GAD67mRNA in dentate gyrus in a rat model of epilepsyrdquo BrainResearch vol 1188 no 1 pp 165ndash172 2008

[31] M L Bullock A J Umen P D Culliton and R T OlanderldquoAcupuncture treatment of alcoholic recidivism a pilot studyrdquoAlcoholism Clinical and Experimental Research vol 11 no 3 pp292ndash295 1987

[32] L ZWu C L Cui J B Tian D Ji and J S Han ldquoSuppression ofmorphine withdrawal by electroacupuncture in rats dynorphinand 120581-opioid receptor implicatedrdquo Brain Research vol 851 no1-2 pp 290ndash296 1999

[33] K Yoshimoto B Kato K Sakai M Shibata T Yano andM Yasuhara ldquoElectroacupuncture stimulation suppresses theincrease in alcohol-drinking behavior in restricted ratsrdquo Alco-holism Clinical and Experimental Research vol 25 no 6supplement pp 63Sndash68S 2001

[34] R J Zhao S S Yoon B H Lee et al ldquoAcupuncture normalizesthe release of accumbal dopamine during thewithdrawal periodand after the ethanol challenge in chronic ethanol-treated ratsrdquoNeuroscience Letters vol 395 no 1 pp 28ndash32 2006

[35] Y Chae C H Yang Y K Kwon et al ldquoAcupuncture attenuatesrepeated nicotine-induced behavioral sensitization and c-Fosexpression in the nucleus accumbens and striatum of the ratrdquoNeuroscience Letters vol 358 no 2 pp 87ndash90 2004

[36] S P Hunt A Pini and G Evan ldquoInduction of c-fos-like proteinin spinal cord neurons following sensory stimulationrdquo Naturevol 328 no 6131 pp 632ndash634 1987

[37] J Buritova V Chapman P Honore and J M Besson ldquoThe con-tribution of peripheral bradykinin B2 receptors to carrageenan-evoked oedema and spinal c-Fos expression in ratsrdquo EuropeanJournal of Pharmacology vol 320 no 1 pp 73ndash80 1997

[38] H Nojima C T Simons J M Cuellar M I Carstens J AMoore and E Carstens ldquoOpioid modulation of scratchingand spinal c-fos expression evoked by intradermal serotoninrdquoJournal of Neuroscience vol 23 no 34 pp 10784ndash10790 2003

[39] H Nojima J M Cuellar C T Simons M I Carstens and ECarstens ldquoSpinal c-fos expression associated with spontaneousbiting in a mouse model of dry skin pruritusrdquo NeuroscienceLetters vol 361 no 1ndash3 pp 79ndash82 2004

[40] R E Coggeshall ldquoFos nociception and the dorsal hornrdquoProgress in Neurobiology vol 77 no 5 pp 299ndash352 2005

[41] M Clark R M Post S R B Weiss and T Nakajima ldquoExpres-sion of c-fos mRNA in acute and kindled cocaine seizures inratsrdquo Brain Research vol 582 no 1 pp 101ndash106 1992

[42] H Z Zhang L X Zhang Y F She W L Li X J Zhangand R J Zhang ldquoEffect of electroacupuncture on apoptosis ofhippocampus tissue in mice with vascular dementiardquo Zhen CiYan Jiu vol 33 no 6 pp 377ndash381 2008

[43] P Fallopa J C Escosteguy-Neto P Varela T N Carvalho AM Tabosa and J G Santos Jr ldquoElectroacupuncture reversesethanol-induced locomotor sensitization and subsequent pERKexpression in micerdquo The International Journal of Neuropsy-chopharmacology vol 15 no 8 pp 1121ndash1133 2012

[44] N Zouhairi S Ba-Mrsquohamed andMBennis ldquoMaternal prenatalstress in rats influences c-fos expression in the spinal cord of theoffspringrdquo Acta Histochemica vol 114 no 5 pp 525ndash533 2012


[45] S Inan N J Dun and A Cowan ldquoNalfurafine prevents 51015840-guanidinonaltrindole- and compound 4880-induced spinal c-fos expression and attenuates 51015840-guanidinonaltrindole-elicitedscratching behavior in micerdquo Neuroscience vol 163 no 1 pp23ndash33 2009

[46] G C Brailoiu S L Dun M Ohsawa et al ldquoKiSS-1 expressionand metastin-like immunoreactivity in the rat brainrdquo Journal ofComparative Neurology vol 481 no 3 pp 314ndash329 2005

[47] E S Lein M J Hawrylycz N Ao et al ldquoGenome-wide atlas ofgene expression in the adult mouse brainrdquo Nature vol 445 no7124 pp 168ndash176 2007

[48] MGasior J T Ungard and JMWitkin ldquoPreclinical evaluationof newly approved and potential antiepileptic drugs againstcocaine-induced seizuresrdquo Journal of Pharmacology and Exper-imental Therapeutics vol 290 no 3 pp 1148ndash1156 1999

[49] R R Matsumoto D L Gilmore B Pouw et al ldquoNovel analogsof the 120590 receptor ligand BD1008 attenuate cocaine-inducedtoxicity in micerdquo European Journal of Pharmacology vol 492no 1 pp 21ndash26 2004

[50] W Vongpatanasin Y Mansour B Chavoshan D Arbique andR G Victor ldquoCocaine stimulates the human cardiovascularsystem via a central mechanism of actionrdquo Circulation vol 100no 5 pp 497ndash502 1999

[51] J M Witkin B Levant A Zapata R Kaminski and M GasiorldquoThe dopamine D

3D2agonist (+)-PD-128907 [(R-(+)-trans-

34a10b-tetrahydro-4-propyl-2H5H-[1]benzopyrano[43-b]-14-oxazin-9-ol)] protects against acute and cocaine-kindledseizures in mice further evidence for the involvement ofD3receptorsrdquo Journal of Pharmacology and Experimental

Therapeutics vol 326 no 3 pp 930ndash938 2008[52] B I Khodorov E G Vornoitskii V B Ignatrsquoeva M R Muku-

mov and G M Kitaıgorodskaia ldquoMechanism of uncoupling ofexcitation and contraction in the myocardial fibres of the frogand guinea pig on blocking the slow sodium-calcium channelswith compound D-600rdquo Biofizika vol 21 no 6 pp 1024ndash10301976

[53] GKWang ldquoCocaine-induced closures of single batrachotoxin-activated Na+ channels in planar lipid bilayersrdquo Journal ofGeneral Physiology vol 92 no 6 pp 747ndash765 1988

[54] M Naguib MM AMagboul A H Samarkandi andM AttialdquoAdverse effects and drug interactions associated with local andregional anaesthesiardquo Drug Safety vol 18 no 4 pp 221ndash2501998

[55] W Lason ldquoNeurochemical and pharmacological aspects ofcocaine-induced seizuresrdquo Polish Journal of Pharmacology vol53 no 1 pp 57ndash60 2001

[56] AMansour and S JWatson ldquoDopamine receptor expression inthe central nervous systemrdquo in PsychopharmacologyThe FourthGeneration of Progress F E Bloom and D J Kupfer Eds pp207ndash219 Raven Press New York NY USA 1995

[57] B Lee S M Han and I Shim ldquoAcupuncture attenuatescocaine-induced expression of behavioral sensitization in ratspossible involvement of the dopaminergic system in the ventraltegmental areardquo Neuroscience Letters vol 449 no 2 pp 128ndash132 2009

[58] L Chen and M Xu ldquoDopamine D1and D

3receptors are

differentially involved in cue-elicited cocaine seekingrdquo Journalof Neurochemistry vol 114 no 2 pp 530ndash541 2010

[59] R L Brackett B Pouw J F Blyden M Nour and R RMatsumoto ldquoPrevention of cocaine-induced convulsions andlethality in mice effectiveness of targeting different sites on the

NMDA receptor complexrdquo Neuropharmacology vol 39 no 3pp 407ndash418 2000

[60] J I Morgan and T Curran ldquoStimulus-transcription couplingin the nervous system involvement of the inducible proto-oncogenes fos and junrdquo Annual Review of Neuroscience vol 14pp 421ndash451 1991

[61] R E Harlan andMMGarcia ldquoDrugs of abuse and immediate-early genes in the forebrainrdquoMolecular Neurobiology vol 16 no3 pp 221ndash267 1998

[62] A M Graybiel R Moratalla and H A Robertson ldquoAmphet-amine and cocaine induce drug-specific activation of the c-fosgene in striosome-matrix compartments and limbic subdivi-sions of the striatumrdquo Proceedings of the National Academy ofSciences of the United States of America vol 87 no 17 pp 6912ndash6916 1990

[63] B Hope B Kosofsky S E Hyman and E J Nestler ldquoRegulationof immediate early gene expression and AP-1 binding in therat nucleus accumbens by chronic cocainerdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 89 no 13 pp 5764ndash5768 1992

[64] S T Young L J Porrino and M J Iadarola ldquoCocaine inducesstriatal c-Fos-immunoreactive proteins via dopaminergic D

1

receptorsrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 88 no 4 pp 1291ndash1295 1991

[65] R C Pierce and L J M J Vanderschuren ldquoKicking the habitthe neural basis of ingrained behaviors in cocaine addictionrdquoNeuroscience and Biobehavioral Reviews vol 35 no 2 pp 212ndash219 2010

[66] H J Groenewegen and H W Berendse ldquoThe specificity of theldquononspecificrdquomidline and intralaminar thalamic nucleirdquoTrendsin Neurosciences vol 17 no 2 pp 52ndash57 1994

[67] C D Young and A Y Deutch ldquoThe effects of thalamic paraven-tricular nucleus lesions on cocaine-induced locomotor activityand sensitizationrdquo Pharmacology Biochemistry and Behaviorvol 60 no 3 pp 753ndash758 1998

[68] S J Spencer J C Fox and T A Day ldquoThalamic paraventricularnucleus lesions facilitate central amygdala neuronal responsesto acute psychological stressrdquo Brain Research vol 997 no 2 pp234ndash237 2004

[69] A E Kelley B A Baldo and W E Pratt ldquoA proposed hypo-thalamic-thalamic-striatal axis for the integration of energybalance arousal and food rewardrdquo Journal of ComparativeNeurology vol 493 no 1 pp 72ndash85 2005

[70] P J Hernandez M E Andrzejewski K Sadeghian J BPanksepp and A E Kelley ldquoAMPAkainate NMDA anddopamine D

1receptor function in the nucleus accumbens core

a context-limited role in the encoding and consolidation ofinstrumental memoryrdquo Learning andMemory vol 12 no 3 pp285ndash295 2005

[71] A S Hamlin K J Clemens E A Choi andG PMcNally ldquoPar-aventricular thalamus mediates context-induced reinstatement(renewal) of extinguished reward seekingrdquo European Journal ofNeuroscience vol 29 no 4 pp 802ndash812 2009

[72] M H James J L Charnley E Jones et al ldquoCocaine- andamphetamine-regulated transcript (CART) signaling withinthe paraventricular thalamus modulates cocaine-seekingbehaviourrdquo PLoS ONE vol 5 no 9 Article ID e12980 2010

[73] S Mraovitch and Y Calando ldquoInteractions between limbicthalamo-striatal-cortical and central autonomic pathways dur-ing epileptic seizure progressionrdquo Journal of Comparative Neu-rology vol 411 no 1 pp 145ndash161 1999


[74] V Neugebauer F Zinebi R Russell J P Gallagher and PShinnick-Gallagher ldquoCocaine and kindling alter the sensitivityof group II and III metabotropic glutamate receptors in thecentral amygdalardquo Journal of Neurophysiology vol 84 no 2 pp759ndash770 2000

[75] D Zhang L Zhang D W Lou Y Nakabeppu J Zhang andM Xu ldquoThe dopamine D

1receptor is a critical mediator for

cocaine-induced gene expressionrdquo Journal of Neurochemistryvol 82 no 6 pp 1453ndash1464 2002

[76] NHiroi J R Brown CNHaile H YeM E Greenberg and EJ Nestler ldquoFosBmutant mice loss of chronic cocaine inductionof Fos-related proteins and heightened sensitivity to cocainersquospsychomotor and rewarding effectsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no19 pp 10397ndash10402 1997

[77] A M Graybiel and R Moratalla ldquoDopamine uptake sites in thestriatum are distributed differentially in striosome and matrixcompartmentsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 86 no 22 pp 9020ndash90241989

[78] M A Medeiros N S Canteras D Suchecki and L E A MMello ldquoc-Fos expression induced by electroacupuncture at theZusanli point in rats submitted to repeated immobilizationrdquoBrazilian Journal of Medical and Biological Research vol 36 no12 pp 1673ndash1684 2003

[79] P KThanosMMichaelides CW Ho et al ldquoThe effects of twohighly selective dopamine D

3receptor antagonists (SB-277011A

and NGB-2904) on food self-administration in a rodent modelof obesityrdquoPharmacology Biochemistry andBehavior vol 89 no4 pp 499ndash507 2008

[80] M C Ritz E J Cone and M J Kuhar ldquoCocaine inhibitionof ligand binding at dopamine norepinephrine and serotonintransporters a structure-activity studyrdquoLife Sciences vol 46 no9 pp 635ndash645 1990

[81] R A Gillis Y M Hernandez H K Erzouki et al ldquoSympatheticnervous system mediated cardiovascular effects of cocaine areprimarily due to a peripheral site of action of the drugrdquo Drugand Alcohol Dependence vol 37 no 3 pp 217ndash230 1995

[82] Z Li K Jiao M Chen and C Wang ldquoEffect of magnitop-uncture on sympathetic and parasympathetic nerve activities inhealthy drivermdashassessment by power spectrumanalysis of heartrate variabilityrdquo European Journal of Applied Physiology vol 88no 4-5 pp 404ndash410 2003

[83] E Haker H Egekvist and P Bjerring ldquoEffect of sensory stim-ulation (acupuncture) on sympathetic and parasympatheticactivities in healthy subjectsrdquo Journal of the Autonomic NervousSystem vol 79 no 1 pp 52ndash59 2000

[84] J S Han X H Chen S L Sun et al ldquoEffect of low- and high-frequency TENS on Met-enkephalin-Arg-Phe and dynorphinA immunoreactivity in human lumbar CSFrdquo Pain vol 47 no 3pp 295ndash298 1991

[85] J S Han X Z Ding and S G Fan ldquoThe frequency as thecardinal determinant for electroacupuncture analgesia to bereversed by opioid antagonistsrdquo Sheng Li Xue Bao vol 38 no5 pp 475ndash482 1986

[86] J S Han ldquoAcupuncture and endorphinsrdquo Neuroscience Lettersvol 361 no 1ndash3 pp 258ndash261 2004

[87] L L Cheng M X Ding C Xiong M Y Zhou Z Y Qiu andQWang ldquoEffects of electroacupuncture of different frequencieson the release profile of endogenous opioid peptides in thecentral nerve system of goatsrdquo Evidence-Based Complementary

and Alternative Medicine vol 2012 Article ID 476457 9 pages2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


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EndocrinologyInternational Journal of



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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


comparedwith other contemporarymedical treatments suchas surgery or drugs [13] Two different strategies are usedin acupuncture therapy manual acupuncture (MA) andelectroacupuncture (EA) EA is amodified formof traditionalMA The advantage of EA is in its combined therapeuticeffects of transcutaneous electric nerve stimulation (TENS)and MA Most studies use EA because the latter can bestandardized by frequency voltage waveform length and soforth [14] Studies on animals and humans have demonstratedthat acupuncture results in multiple biological responses [1516] The best characterized mechanism is via the endogenousopioid peptides and their receptors [17]

13 Treating Epilepsy with Acupuncture Acupuncture hasbeen used to treat epilepsy in China for thousands of yearsMany reports have suggested that acupuncture TENS andother alternative therapies may produce positive effects inepilepsy [19] although some studies disagree [20]Themajoradvantage of acupuncture is the absence of side effectsIn the clinic acupuncture at specific acupoints such asthe Zusanli (ST36) Dazhui (GV14) and Baihui (GV20)has significantly ameliorated symptoms of epilepsy [21ndash23]Although some evidence supports this contention [24ndash26]the precise mechanism remains unknown It is noted thatacupuncture to GV20 point has been used to treat loss ofconsciousness and tinnitus in addition to relief of mentalabstraction sluggish speech and hysteria [27] Recent studieshave shown that EA inhibits seizures in experimental ratmodels These protective effects may be related to increasedconcentrations of inhibitory amino acids [28] decreasedlevels of nitric oxide in the central nervous system (CNS)[26 29] or increased cellular glutamic acid decarboxylase-67 mRNA expression thereby increasing the production of120574-aminobutyric acid (GABA) [30] The possibility that EAprotects against cocaine-induced seizures and death has notyet been explored

14 Acupuncture and Abused Drugs Acupuncture has beenwidely used throughout Asian countries for treating manyfunctional disorders such as substance abuse and mental ill-ness [31] Some preclinical data have shown that acupuncturecanmodify themorphinewithdrawal syndrome and suppressalcohol drinking behavior in rats [32 33] Further evidencesuggests that acupuncture at a specific acupoint (ShenmenHT7) attenuates the ethanol-induced dopamine release in thenucleus accumbens through the GABAB receptor [34] andsuppresses c-Fos expression in the nucleus accumbens andventromedial striatum following a nicotine challenge in ratssensitized to nicotine [35]

15 c-Fos Expression Expression of c-fos gene or Fos proteinis widely used as a marker to identify neuronal pathwaysinvolved in the integration of noxious inputs [36ndash40] Highdoses of cocaine have been reported to induce c-fos mRNAexpression in many brain regions [41]

In this study EA experimentswere conducted to (1) estab-lish the applicability of EA to cocaine-induced seizure and

mortality in a mouse model (2) determine the frequency-dependent antiseizure activity of EA and (3) correlate theefficacy of EA with the expression of Fos protein in the brain

2 Methods

21 Laboratory Animals Male ICR mice (28ndash35 g BioLascoTaiwan Co Ltd Taiwan) were housed under a 1212 hlightdark cycle with food and water available ad libitum inour animal facility for at least 4 days prior to the experimentswhich were conducted between 1000 and 1700 hThe exper-imental procedures were approved by the China MedicalUniversity Institutional Animal Care and Use Committee inaccordance with the Chinese Taipei Society of LaboratoryAnimal Sciences guidance on care and use of laboratoryanimals Experiments were designed to keep the number ofmice at aminimumand care was taken tominimize suffering

22 Electroacupuncture For the EA treatment animals werecovered by paraffin film and restrained by tapes withoutanesthesia A pair of stainless steel acupuncture needles(Tianjin HaingLimSou Won Medical CO Ltd Gauge 40)were inserted 3-4mm deep into the murine equivalent ofthe human GV14 and GV20 that is (1) the skin at thelocation between the last cervical and the first thoracicvertebral spinous processes at the midline of the back whichis equivalent to the human GV14 and (2) at the vertex ofthe parietal bone that is the midpoint of the connectingline between the auricular apices which is equivalent to thehuman GV20 acupoints [42 43] EA stimuli were deliveredby an EA Trio 300 stimulator (Ito Japan) at 1mA intensitiesfor a 15min duration at a frequency of 2 50 or 100Hz witha pulse width of 150 120583sThe two electrodes were connected tothe needles which were inserted into GV20 and GV14 In thecontrol group animals were also covered by paraffin film andrestrained by tape for 15min [44] A sham EAwas performedby bilateral insertion of a pair of stainless steel acupunctureneedles approximately 3-4mm deep into the middle of eachscapula which is equivalent to the human Tianzong (SI11)

23 Behavioral Study Following the injection of cocaineor saline animals were placed in a plastic observation boxand seizure scores were assessed by the Itzhak scale whichcategorizes five stages according to their severity over a30min period [18]The Itzhak stages of seizure are as follows

Stage 1 Normal behavior (moving about the cage sniffingand rearing)

Stage 2 Hyperactivity (running movement characterized byrapid changes in position)

Stage 3 Animal remains in the same place for several secondswith fast repetitive movements of the head face mouth orforelimb as well as head nodding

Stage 4 Forelimb clonus and rearing






2O2




3receptor [51]


3 Results







(a) (b)

∙ GV20

∙ GV14

SI11∙

SI11∙

(c)


5

4

3

2

1

0

lowastlowast lowast

Cocaine 75mgkg

Seiz

ure s

core

s


119899 = 21

119899 = 20 119899 = 20 119899 = 23 119899 = 20

needleinsertion

EA2 Hz

EA50 Hz

EA100 Hz

shamEA

119899 = 20




lowast lowast

lowastlowastlowast

lowastlowast

80

60

40

20

0

Cocaine 125 mgkgM

orta

lity

()


119899 = 34

119899 = 18 119899 = 18 119899 = 32 119899 = 18 119899 = 17

needleinsertion

EA2 Hz

EA50 Hz

EA100 Hz

shamEA





orta

lity

()

100

80

60

40

20


+ +

EA 2 Hz EA 50 Hz119899 = 20

119899 = 21 119899 = 20

Procaine 400 mgkg














(a)


0

10

20

30

40

50

Num

ber o

f c-F

os p

ositi

ve n

ucle

i



lowast

(b)





3mRNA







(a)

60

50

40

30

20

10

0

lowastlowastlowast


Amygdala

Num

ber o

f c-F

os p

ositi

ve n

ucle

i


(b)






4 Discussion




(a)


0

10

20

30

40

50

60

Num

ber o

f c-F

os p

ositi

ve n

ucle

i

lowastlowastlowast


lowastlowastlowast

Caudoputamen

(b)








119899 = 11

119899 = 10

119899 = 12

Cocaine (75 mgkg)

119899 = 9

6

5

4

3

2

1

0

Seiz

ure s

core

s

lowast lowast

lowast

CocaineCocaine+

EA

Cocaine+

EA+

SB-277011-A

Cocaine+

SB-277011-A






3mRNA [56] The















3receptor-deficient






3receptorsmediate

















Acknowledgments


References

































































3receptors are









1






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















2O2




3receptor [51]


3 Results







(a) (b)

∙ GV20

∙ GV14

SI11∙

SI11∙

(c)


5

4

3

2

1

0

lowastlowast lowast

Cocaine 75mgkg

Seiz

ure s

core

s


119899 = 21

119899 = 20 119899 = 20 119899 = 23 119899 = 20

needleinsertion

EA2 Hz

EA50 Hz

EA100 Hz

shamEA

119899 = 20




lowast lowast

lowastlowastlowast

lowastlowast

80

60

40

20

0

Cocaine 125 mgkgM

orta

lity

()


119899 = 34

119899 = 18 119899 = 18 119899 = 32 119899 = 18 119899 = 17

needleinsertion

EA2 Hz

EA50 Hz

EA100 Hz

shamEA





orta

lity

()

100

80

60

40

20


+ +

EA 2 Hz EA 50 Hz119899 = 20

119899 = 21 119899 = 20

Procaine 400 mgkg














(a)


0

10

20

30

40

50

Num

ber o

f c-F

os p

ositi

ve n

ucle

i



lowast

(b)





3mRNA







(a)

60

50

40

30

20

10

0

lowastlowastlowast


Amygdala

Num

ber o

f c-F

os p

ositi

ve n

ucle

i


(b)






4 Discussion




(a)


0

10

20

30

40

50

60

Num

ber o

f c-F

os p

ositi

ve n

ucle

i

lowastlowastlowast


lowastlowastlowast

Caudoputamen

(b)








119899 = 11

119899 = 10

119899 = 12

Cocaine (75 mgkg)

119899 = 9

6

5

4

3

2

1

0

Seiz

ure s

core

s

lowast lowast

lowast

CocaineCocaine+

EA

Cocaine+

EA+

SB-277011-A

Cocaine+

SB-277011-A






3mRNA [56] The















3receptor-deficient






3receptorsmediate

















Acknowledgments


References

































































3receptors are









1






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

∙ GV20

∙ GV14

SI11∙

SI11∙

(c)


5

4

3

2

1

0

lowastlowast lowast

Cocaine 75mgkg

Seiz

ure s

core

s


119899 = 21

119899 = 20 119899 = 20 119899 = 23 119899 = 20

needleinsertion

EA2 Hz

EA50 Hz

EA100 Hz

shamEA

119899 = 20




lowast lowast

lowastlowastlowast

lowastlowast

80

60

40

20

0

Cocaine 125 mgkgM

orta

lity

()


119899 = 34

119899 = 18 119899 = 18 119899 = 32 119899 = 18 119899 = 17

needleinsertion

EA2 Hz

EA50 Hz

EA100 Hz

shamEA





orta

lity

()

100

80

60

40

20


+ +

EA 2 Hz EA 50 Hz119899 = 20

119899 = 21 119899 = 20

Procaine 400 mgkg














(a)


0

10

20

30

40

50

Num

ber o

f c-F

os p

ositi

ve n

ucle

i



lowast

(b)





3mRNA







(a)

60

50

40

30

20

10

0

lowastlowastlowast


Amygdala

Num

ber o

f c-F

os p

ositi

ve n

ucle

i


(b)






4 Discussion




(a)


0

10

20

30

40

50

60

Num

ber o

f c-F

os p

ositi

ve n

ucle

i

lowastlowastlowast


lowastlowastlowast

Caudoputamen

(b)








119899 = 11

119899 = 10

119899 = 12

Cocaine (75 mgkg)

119899 = 9

6

5

4

3

2

1

0

Seiz

ure s

core

s

lowast lowast

lowast

CocaineCocaine+

EA

Cocaine+

EA+

SB-277011-A

Cocaine+

SB-277011-A






3mRNA [56] The















3receptor-deficient






3receptorsmediate

















Acknowledgments


References

































































3receptors are









1






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















orta

lity

()

100

80

60

40

20


+ +

EA 2 Hz EA 50 Hz119899 = 20

119899 = 21 119899 = 20

Procaine 400 mgkg














(a)


0

10

20

30

40

50

Num

ber o

f c-F

os p

ositi

ve n

ucle

i



lowast

(b)





3mRNA







(a)

60

50

40

30

20

10

0

lowastlowastlowast


Amygdala

Num

ber o

f c-F

os p

ositi

ve n

ucle

i


(b)






4 Discussion




(a)


0

10

20

30

40

50

60

Num

ber o

f c-F

os p

ositi

ve n

ucle

i

lowastlowastlowast


lowastlowastlowast

Caudoputamen

(b)








119899 = 11

119899 = 10

119899 = 12

Cocaine (75 mgkg)

119899 = 9

6

5

4

3

2

1

0

Seiz

ure s

core

s

lowast lowast

lowast

CocaineCocaine+

EA

Cocaine+

EA+

SB-277011-A

Cocaine+

SB-277011-A






3mRNA [56] The















3receptor-deficient






3receptorsmediate

















Acknowledgments


References

































































3receptors are









1






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)


0

10

20

30

40

50

Num

ber o

f c-F

os p

ositi

ve n

ucle

i



lowast

(b)





3mRNA







(a)

60

50

40

30

20

10

0

lowastlowastlowast


Amygdala

Num

ber o

f c-F

os p

ositi

ve n

ucle

i


(b)






4 Discussion




(a)


0

10

20

30

40

50

60

Num

ber o

f c-F

os p

ositi

ve n

ucle

i

lowastlowastlowast


lowastlowastlowast

Caudoputamen

(b)








119899 = 11

119899 = 10

119899 = 12

Cocaine (75 mgkg)

119899 = 9

6

5

4

3

2

1

0

Seiz

ure s

core

s

lowast lowast

lowast

CocaineCocaine+

EA

Cocaine+

EA+

SB-277011-A

Cocaine+

SB-277011-A






3mRNA [56] The















3receptor-deficient






3receptorsmediate

















Acknowledgments


References

































































3receptors are









1






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)

60

50

40

30

20

10

0

lowastlowastlowast


Amygdala

Num

ber o

f c-F

os p

ositi

ve n

ucle

i


(b)






4 Discussion




(a)


0

10

20

30

40

50

60

Num

ber o

f c-F

os p

ositi

ve n

ucle

i

lowastlowastlowast


lowastlowastlowast

Caudoputamen

(b)








119899 = 11

119899 = 10

119899 = 12

Cocaine (75 mgkg)

119899 = 9

6

5

4

3

2

1

0

Seiz

ure s

core

s

lowast lowast

lowast

CocaineCocaine+

EA

Cocaine+

EA+

SB-277011-A

Cocaine+

SB-277011-A






3mRNA [56] The















3receptor-deficient






3receptorsmediate

















Acknowledgments


References

































































3receptors are









1






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)


0

10

20

30

40

50

60

Num

ber o

f c-F

os p

ositi

ve n

ucle

i

lowastlowastlowast


lowastlowastlowast

Caudoputamen

(b)








119899 = 11

119899 = 10

119899 = 12

Cocaine (75 mgkg)

119899 = 9

6

5

4

3

2

1

0

Seiz

ure s

core

s

lowast lowast

lowast

CocaineCocaine+

EA

Cocaine+

EA+

SB-277011-A

Cocaine+

SB-277011-A






3mRNA [56] The















3receptor-deficient






3receptorsmediate

















Acknowledgments


References

































































3receptors are









1






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















119899 = 11

119899 = 10

119899 = 12

Cocaine (75 mgkg)

119899 = 9

6

5

4

3

2

1

0

Seiz

ure s

core

s

lowast lowast

lowast

CocaineCocaine+

EA

Cocaine+

EA+

SB-277011-A

Cocaine+

SB-277011-A






3mRNA [56] The















3receptor-deficient






3receptorsmediate

















Acknowledgments


References

































































3receptors are









1






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































Acknowledgments


References

































































3receptors are









1






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





































































3receptors are









1






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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