Endogenous Opiates in the Nucleus Tractus Solitarius Mediate Electroacupuncture-Induced Sleep ...

12 Pages·2014·1.78 MB·English

HindawiPublishingCorporation
Evidence-BasedComplementaryandAlternativeMedicine
Volume2011,ArticleID159209,11pages
doi:10.1093/ecam/nep132
Original Article
Endogenous Opiates in the Nucleus Tractus Solitarius Mediate
Electroacupuncture-Induced Sleep Activities in Rats
Chiung-HsiangCheng,1Pei-LuYi,2Jaung-GengLin,3andFang-ChiaChang1,3
1DepartmentofVeterinaryMedicine,SchoolofVeterinaryMedicine,NationalTaiwanUniversity,Taipei106,Taiwan
2DepartmentofMedicalTechnology,Jen-TehJuniorCollegeofMedicine,NursingandManagement,Miaoli,Taiwan
3GraduateInstituteofAcupunctureScience,CollegeofChineseMedicine,ChinaMedicalUniversity,Taichung,Taiwan
CorrespondenceshouldbeaddressedtoFang-ChiaChang,[email protected]
Received26March2009;Accepted6August2009
Copyright©2011Chiung-HsiangChengetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttribution
License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly
cited.
Electroacupuncture (EA) possesses various therapeutic effects, including alleviation of pain, reduction of inflammation and
improvement of sleep disturbance. The mechanisms of EA on sleep improvement, however, remain to be determined. It has
beenstatedinancientChineseliteraturethattheAnmian(EX17)acupointisoneofthetriggerpointsthatalleviatesinsomnia.
We previously demonstrated that EA stimulation of Anmian acupoints in rats during the dark period enhances non-rapid
eye movement (NREM) sleep, which involves the induction of cholinergic activity in the nucleus tractus solitarius (NTS). In
addition to cholinergic activation of the NTS, activation of the endogenous opioidergic system may also be a mechanism by
whichacupunctureaffectssleep.Therefore,thisstudywasdesignedtoinvestigatetheinvolvementoftheNTSopioidergicsystem
in EA-induced alterations in sleep. Our present results indicate that EA of Anmian acupoints increased NREM sleep, but not
rapid eye movement sleep, during the dark period in rats. This enhancement in NREM sleep was dose-dependently blocked
by microinjection of opioid receptor antagonist, naloxone, and the μ-opioid receptor antagonist, naloxonazine, into the NTS;
administrationsofδ-receptorantagonist,natrindole,andtheκ-receptorantagonist,nor-binaltrophimine,however,didnotaffect
EA-inducedalterationsinsleep.Furthermore,β-endorphinwassignificantlyincreasedinboththebrainstemandhippocampus
aftertheEAstimuli,aneffectblockedbyadministrationofthemuscarinicantagonistscopolamineintotheNTS.Ourfindings
suggestthatmechanismsofEA-inducedNREMsleepenhancementmaybemediated,inpart,bycholinergicactivation,stimulation
oftheopiodergicneuronstoincreasetheconcentrationsofβ-endorphinandtheinvolvementoftheμ-opioidreceptors.
1.Introduction Zusanli (ST36), Taichong (LR3), Baihui (DU20), Dazhui
(DU40), Tainzhu (BL10), Bishu (BL20) and Zhongwan
Electroacupuncture (EA), which consists of passing a con- (RN12)[3,4].
tinuous electric current through needles inserted into the The mechanisms by which EA functions to alleviate
acupoints to obtain the therapeutic effects, that is, allevia- clinicalsymptomsremainlargelyunknown,althoughappli-
tion of pain, reduction of inflammation and management cationsofEAhavebeenwidelydescribedintheChineseliter-
of insomnia, is modified from the traditional Chinese ature.Thespinalgate-controltheory[5]andtheactivationof
acupuncture. Insomnia is one of the most common sleep centralendorphinand/ormonoaminergicsystems(i.e.,sero-
disordersandithasbeendemonstratedthattheeffectiveness tonin and norepinephrine) [6] have been hypothesized in
rate of acupuncture for relieving insomnia is about 90% mediating the EA-induced analgesia. In addition, acupunc-
[1, 2]. Several specific acupoints have been identified for turemayreducetheinflammation-inducedelevationofbody
insomniatreatmentbaseduponthedifferentiationandsigns temperature by suppressing hypothalamic production of
of symptoms according to traditional Chinese medicine. pro-inflammatorycytokines[7].Thecentralopioidergicand
Among the acupoints used, Shenmen (HT7), Sanyinjiao serotonergic systems also mediate the suppressive effects of
(SP6)andAnmian(EX17)arethemostcommon,although acupunctureoncapsaicin-inducedneurogenicinflammation
other acupoints may also be used, such as Neiguan (PC6), [8].Recentfindingssuggestthattheinductionofvagusnerve
2 Evidence-BasedComplementaryandAlternativeMedicine
activityappearstobeanothersignificantfactorformediating 2.2. Animals. Male Sprague-Dawley rats (250–300g;
theactionofacupuncture[9,10].Thecaudalnucleustractus NationalLaboratoryAnimalBreedingandResearchCenter,
solitarius (NTS) may be activated by acupuncture, since Taiwan) were used in this study. Rats were anesthetized by
NTS is located in the dorsomedial medulla oblongata and intraperitoneal injection with ketamine/xylazine (87/
receives afferents primarily from the vagus and glossopha- 13mgkg−1) and were given an analgesic (1mg/rat
ryngeal nerves [11]. Ascending projections from the NTS morphine) and an antibiotic (5000IU/rat penicillin G ben-
are traced through the lateral and dorsal tegmentum and zathine) to reduce pain and avoid infection. Rats were
periventriculargrayuptotherostralponsandmidbrain,and surgically implanted with three electroencephalogram
terminateintheparabrachialnucleus,whichinturnprojects (EEG) screw electrodes as earlier described [19] and the
tothethalamus,hypothalamus,preopticarea,bednucleusof microinjection guide cannulae directed into the NTS (AP,
thestriaterminalis,amygdalaandthefrontalcortex,regions 13.30mm from bregma; ML, 1.2mm and DV, 8.2mm
commonly belonging to the visceral-limbic forebrain [12, relative to bregma). The coordinates were adopted from
13].Fromtheseanatomicaldata,itdoesnotappearthatthe the Paxinos and Waton rat altas [20]. Two unilateral screw
predominanteffectoftheNTSisviathereticularactivating EEG electrodes were placed over the right hemisphere of
system but instead is via limbic forebrain structures, which the frontal and parietal cortices and a third EEG electrode
areimplicatedinthesleepregulation.Furthermore,thelow- was placed over the cerebellum and served to ground the
frequency electrical stimulation of the medullary reticular animal to reduce signal artifacts. Insulated leads from
formation,particularlythedorsalreticularformationandthe EEG electrodes were routed to a Teflon pedestal (Plastics
caudalNTS,producescorticalsynchronizationindicativeof One, Roanoke, VA, USA). The Teflon pedestal was then
slow-wavesleep(SWS)inanawakeanimal[14].Conversely, cemented to the skull with dental acrylic (Tempron, GC
lesions of the dorsal reticular formation and of the NTS Co., Tokyo, Japan). The incision was treated topically with
produceddesynchronizationoftheEEGinasleepinganimal polysporin (polymixin B sulfate—bacitracin zinc) and the
[15]. These results all suggest the existence of neurons in animals were allowed to recover for 7 days prior to the
theNTSthatareinvolvedingeneratingsleep.Furthermore, initiationofexperiments.Theratswerehousedseparatelyin
microinjection of morphine into the NTS provokes an individualrecordingcagesintheisolatedroom,inwhichthe
enhancement of SWS and this effect is blocked by nalox- temperaturewasmaintainedat23 ± 1◦Candthelight:dark
one [16], suggesting the somnogenic effect of opioidergic rhythmwascontrolledina12:12hcycle(40Watt×4tubes
systemintheNTS.Ourpreviousobservationsdemonstrate illumination). Food (5001 rodent diet, LabDiet) and water
that activation of cholinergic system in the caudal NTS were available ad libitum. All procedures performed in this
of the medulla oblongata mediates the enhancement of study were approved by the National Taiwan University
non-rapid eye movement (NREM) sleep induced by EA AnimalCareandUseCommittee.
stimulationofAnmian(EX17)acupoints[17].Nonetheless,
EA may also increase β-endorphin concentrations in the
NTS, which subsequently alter sleep, because the NTS area 2.3. Experimental Protocol. On the second postsurgical day,
is one of the anatomically distinct β-endorphin pathways the rats were connected to the recording apparatus via a
in the brain influenced by EA [18]. Therefore, this study flexible tether. As such, the rats were allowed relatively
was designed to further clarify whether the activation of unrestrictedmovementwithintheirowncages.Threegroups
cholinergic system in the NTS after EA stimuli enhances ofratswereusedinthestudyasfollows:Group1(n=8)was
endogenous opioidergic activity and what type(s) of opi- used to determine the effects of opioid receptor antagonist
oid receptors are involved in EA-induced alterations in (naloxone)andμ-receptorantagonist(naloxonazine)onEA-
sleep. induced alterations in sleep; Group 2 (n = 8) was used to
elucidate the effects of δ-receptor antagonist (naltrindole)
and κ-receptor antagonist (nor-binaltorphimine) on EA-
2.Methods induced alterations in sleep; Group 3 (n = 24) was used
to depict the action of NTS muscarinic receptors on EA-
2.1. Pharmacological Agents. Stock solutions of muscarinic induced β-endorphin expression (determined by enzyme-
receptor antagonist, scopolamine hydrobromide (Sigma, St linked immunosorbent assay (ELISA)) after microinjection
Louis, MO, USA), a broad-spectrum opioid antagonist of scopolamine into the NTS. One week after rats had
(naloxonehydrochloride(Tocris,Bristol,UK)),aμ-receptor adaptedtothe12:12hlight:darkcycleaftersurgery,a23-
antagonist (naloxonazine dihydrochloride (Tocris)), a δ- hundisturbedbaselinerecordingswereobtainedbeginning
receptorantagonist(naltrindolehydrochloride(Tocris))and at dark onset on the first recording day in rats in Groups 1
a κ-receptor antagonist (nor-binaltorphimine dihydrochlo- and 2. When EA was given (see later), all rats were lightly
ride (Tocris)) were dissolved in pyrogen-free saline (PFS). anesthetizedwithone-thirdofthedoseofketamine/xylazine
Thestocksolutionswerestoredat4◦Cuntiluse.Thedoseof used in the surgery, after which rat wake-up time is 20–
scopolamineusedwas20μgμL−1,whereasnaloxone,nalox- 25min.A20-minperiodofEAstimulationwasadministered
onazine,naltrindoleandnor-binaltorphimineweremicroin- beforetheonsetofthedarkperiod.Theanesthetizationwas
jected at three different doses, 0.1, 1.0 and 10μgμL−1. given 25min prior to the dark period onset and lasted for
The total volume used for each microinjection was 20min.Therationaleforcarryingouttheexperimentinthe
1μL. darkness is that rats are active with a lowest level of sleep
Evidence-BasedComplementaryandAlternativeMedicine 3
during the dark period, and a manipulation, if it possesses striatum, were dissected and frozen in −80◦C until assay.
ability to increase sleep, would significantly augment sleep Thesetissueswereusedforβ-endorphinELISAasdescribed
during the dark period. In contrast, it may not be easy to inthefollowing.
enhancesleepduringthelightperiodwhensleepactivityis
atitshighestcircadianlevel.Sinceweexpectedtofindasleep
2.4. Apparatus and Recording. Signals from the EEG elec-
enhancementaftertheEAstimuliattheAnmian(EX17),we
trodes were fed into an amplifier (Colbourn Instruments,
therefore manipulated the EA stimulation before the onset
Lehigh Valley, PA; model V75-01). The EEG was amplified
of the dark period and analyzed the sleep alteration during
(factor of 5000) and analog bandpass filtered between 0.1
the subsequent dark period. The rats in group 1 were both and 40Hz (frequency response: ±3dB; filter frequency roll
administered PFS intraperitoneally (IP) and microinjected off:12dB/octave).Grossbodymovementsweredetectedby
intotheNTS( PFS+PFS)at25minpriortothedarkonset
ip custom-made infrared-based motion detectors (Biobserve
on two consecutive days, and recordings obtained for 24-h
GmbH,Germany)andthemovementactivitywasconverted
beginningafterthesecondinjection.Theeffectsofanesthesia
to a voltage output which was digitized and integrated into
withtheNTSmicroinjectionofPFS( ketamine+PFS)on
ip 1-s bins. These conditioned signals (EEGs and gross body
sleepweredeterminedafterIPinjectionofketamine/xylazine
movements) were subjected to analog-to-digital conversion
and the NTS PFS microinjection on two consecutive days.
with16-bitprecisionatasamplingrateof128Hz(NIPCI-
A sham EA ( ketamine + PFS + sham EA) was delivered
ip 6033E; National Instruments, Austin, TX). The digitized
to control for the non-specific effect of the electrical stim-
EEG waveform and integrated values for body movement
ulation,althoughourpreviousstudyhadconfirmedthatno
were stored as binary computer files pending subsequent
non-specificeffectwasobservedaftertheshamEA[17].The
analyses.
EA stimuli under anesthesia ( ketamine + PFS + EA) were
ip Postacquisition determination of the vigilance state was
alsoperformedbeforethedarkonsetontwoconsecutivedays
donebyvisualscoringof12-sepochsusingcustomsoftware
and sleep-wake behavior after the second EA stimulation
(ICELUS, M. R. Opp) written in LabView for Windows
was then determined. Subsequently, three different doses
(NationalInstruments).Theanimal’sbehaviorwasclassified
(0.1, 1.0 and 10μg) of naloxone ( ketamine + naloxone +
ip as SWS, rapid eye movement sleep (REMS) or waking
EA) and naloxonazine ( ketamine + naloxonazine + EA)
ip based on previously defined criteria [19]. Briefly, SWS is
were administered 25min prior to the dark onset on the
characterized by large-amplitude EEG slow waves, high
seconddayofEAstimulationandthe24-hsleeppatternwas
power density values in the delta frequency band (0.5–
determined.Atleast1daywithoutinjectionswasscheduled
4Hz) and lack of gross body movements. During REMS,
betweeneachmanipulation.TheEAstimuluswasdelivered
theamplitudeoftheEEGisreduced,thepredominantEEG
via the bilateral insertion of stainless needles (32 gauge ×
power density occurs within the theta frequency (6–90Hz)
1(cid:3)(cid:3), Shanghai Yanglong Medical Articles Co.) on Anmian
andtherearephasicbodytwitches.Duringwaking,therats
(EX17)pointsinthedepthof2mm.Thestimulusconsisted
are generally active. There are protracted body movements.
ofatrainofbiphasicpulses(150μsdurationeach)of10Hz
TheamplitudeoftheEEGissimilartothatobservedduring
with intensity of 3mA, and was delivered by Functions
REMS,butpowerdensityvaluesinthedeltafrequencyband
ElectricalStimulator(Trio300,I.T.O.,Japan).Theacupoint
aregenerallygreaterthanthoseinthetafrequencyband.In
“Anmian(EX17)”islocatedatmidpointbetweenYifeng(TH
additiontotheamountoftimespentineachvigilancestate,
17) and Fengchi (GB 20); Yifeng (TH 17) locates posterior
the number and duration of individual bouts of behaviors
to the lobule of the ear in the depression between the
weredeterminedusingcriteriamodifiedfromthoseofTobler
mandibleandmastoidprocessandFengchi(GB20)locates
andcolleagues[21,22],asdescribedearlier[19].
in the depression between the upper portion of musculus
sternocleidomastoideus and musculus trapezius in human.
2.5. ELISA for β-Endorphin. Rat β-endorphin ELISA
The location of Anmian (EX17) in rats is at the relative
kits were obtained from Phoenix Pharmaceuticals Inc.
anatomical location between the strenocleidomastoideus
(Burlingame, CA, USA) and the procedures followed the
muscle and the splenius capitis muscle, as in the human
standard instructions provided by the manufacturers.
acupointmap.ShamEAwasperformedbystimulationofa
Absorbance was measured by an ELISA plate reader
non-acupointlocatedattheventralconjunctionbetweenthe
(Multiskan EX, Thermo Electron Corp., Waltham, MA,
forelimbandthetrunkasdescribedearlier[17].Thoserats
USA) with the wavelength set at 450 and 550nm. The
in group 2 underwent a similar protocol to those in group
sensitivity is 0.21ngmL−1, the assay range is between 0.21
1, except that the substances administered were naltrindole
and 3.15ngmL−1, the intra-assay coefficient of variation
( ketamine + naltrindole + EA) and nor-binaltorphimine
ip is <5% and the inter-assay coefficient of variation is
( ketamine + nor-binaltorphimine + EA). Rats in group 3
ip
<14%. There is no cross-reactivity for met-enkephalin and
weredividedintofoursubgroupsandreceived ketamine+
ip
leu-enkephalin.
PFS, ketamine + PFS + sham EA, ketamine + PFS + EA
ip ip
and ketamine + scopolamine + EA, respectively. Microin-
ip
jectionofscopolamineintotheNTSwasadministeredprior 2.6. Statistical Analyses for Experiment Protocol. All values
to the second EA stimulation. Rats were then decapitated acquired from sleep-wake recording were presented as the
at the dark onset and five distinct brain regions, including mean ± SEMfortheindicatedsamplesizes.One-wayanaly-
the hypothalamus, cortex, brainstem, hippocampus and sesofvariance(ANOVA)forthedurationofeachvigilance
4 Evidence-BasedComplementaryandAlternativeMedicine
state (SWS, REMS, WAKE) and for sleep architecture naloxonazine altered REM sleep (Figures 2(c) and 2(d)).
parameters were performed, comparing before and after A mirror effect on the EA-induced decrease of waking was
manipulationwithinsubjects,acrossacertainoftimeblock. also been observed for both naloxone and naloxonazine
An α level of P ≤ .05 was taken as indicating a statistically (Figures2(e)and2(f)).
significant difference. If statistically significant differences Analysis of sleep architecture parameters across 1–12h
were detected, a Scheffe post hoc comparison was made during the dark period revealed that the effect of ketamine
to determine which hourly intervals during experimental onthesuppressionofNREMandREMsleepwasprimarily
conditions deviated from values obtained from the same due to an increase in episode duration of waking, although
animalsduringcontrolconditions.Thestatisticalevaluation therewasatendencyfordecreasesinbothREMsleepepisode
fortheβ-endorphinELISAusedanunpairedStudent’st-test number and in NREM sleep episode duration (Table1).
comparingtheaveragesbetweenthegroupsanalyzed. TheincreaseofNREMsleepafterEAstimuliwasprimarily
because of the increase in the duration of a single episode
(Table1),whichissimilartoourpreviousresult[17].Effects
3.Results of naloxone and naloxonazine on blocking EA-induced
enhancementofNREMsleepweremediatedbyreversingthe
3.1. Naloxone and Naloxonazine Blocked the EA-Induced EA-inducedaugmentationofNREMsleepepisodeduration
Alterations in Sleep. Anesthetization of rats for 25min (Table1).
withketamine/xylazinepriortothedarkperiodsuppressed
both NREM and REM sleep for the first 4h of the dark 3.2.NaltrindoleandNor-BinaltorphimineDidNotAffectEA-
period. The time spent in NREM sleep during the first 4-
Induced Alterations in Sleep. Administration of either the
h period after ketamine + PFS decreased to 10.8 ± 2.5%
ip δ-opioid receptor antagonist naltrindole or the κ-receptor
from 22.4 ± 2.5% acquired after PFS + PFS (F =
ip (1,62) antagonist nor-binaltorphimine prior to EA stimulation
10.711, P = .002, Figures 1(a) and 1(b)). REM sleep was did not affect EA-induced alterations in sleep (Figure3).
suppressed from 6.5 ± 1.8% obtained after PFS + PFS
ip Similarly,theseantagonistsdidnotalteranyaspectofsleep
to 0.8 ± 0.3% acquired after ketamine + PFS (F =
ip (1,62) architecture(datanotshown).
8.697, P = .005, Figures 1(c) and 1(d)). A concomitant
enhancement of wakefulness was observed during the first
3.3. Scopolamine Suppressed EA-Induced Expression of
4hafterreceivingketamineanesthetization(Figures1(e)and
Endogenous β-Endorphin. Our result demonstrated that
1(f)).ApplicationofshamEAdidnotalteranyaspectofsleep
deliveryofshamEAstimulididnotaltertheconcentrations
parameters (data not shown), which is similar to our pre-
of β-endorphin within five distinct brain structures,
viousobservation[17].Twenty-minuteEAstimulidelivered
including the brainstem, hypothalamus, cortex, hippo-
beforethedarkperiodontwoconsecutivedayssignificantly
campusandstriatum(Figure4).However,applicationofEA
augmented NREM sleep during post-manipulation 5–8h.
stimuliatAnmian(EX17)acupointsfor20minsignificantly
The percentage of time spent in NREM sleep during 5–
8h increased from 18.3 ± 2.7% obtained after ketamine enhanced the levels of β-endorphin in the brainstem and
ip
+ PFS to 34.0 ± 3.8% after vketamine + PFS + EA (F hippocampus, but not in the cortex, hypothalamus and
(1,62)
= 9.180, P = .004, Figures 1(a) and 1(b)). Analysis of striatum (Figure4). Administration of the muscarinic
receptor antagonist scopolamine directly into the NTS
12-h dark period revealed that NREM sleep was enhanced
from 14.8 ± 1.5% after ketamine + PFS to 21.7 ± 2.1% before the EA stimuli blocked EA-induced increases of
ip
after ketamine + PFS + EA (F = 6.923, P = .010). β-endorphininthebrainstemandhippocampus(Figure4).
ip (1,190)
REM sleep was not significantly altered by EA. A decrease
ofwakingattheexpenseofSWSwasobservedduring5–8h 4.Discussion
(Figures1(e)and1(f)).
Administration of three different doses (0.1, 1.0 and Insomnia is a common sleep complaint among elderly
10.0μg) of naloxone, a broad-spectrum opioid antagonist, peopleandyoungadults,whichmayresultfrompsychiatric
into the NTS dose-dependently blocked EA-induced illness, sociopsychological stress, a medical problem, poor
increases of NREM sleep, especially during post- sleep habits or a primary sleep disorder. Epidemiological
manipulation 5–8h (Figures 2(a) and 2(b)). Across the surveys have shown that 10%–20% of adults have suffered
entire 12-h dark period recording, NREM sleep was from moderate to severe insomnia [23], although the
suppressed from 21.7 ± 2.1% after ketamine + PFS + percentageislowerthanthatof40%–70%ofhealthyelderly
ip
EA to 13.4 ± 2.1% after ketamine + naloxone (10.0μg) people who suffer from chronic sleep disturbances [24].
ip
+ EA (F = 7.631, P = .007, Figures 2(a) and 2(b)). Sedative-hypnotic medications, including benzodiazepines
(1,190)
Administration of μ-opioid receptor antagonist, naloxon- andnon-benzodiazepines,arethemostcommontreatments
azine, also exhibited a dose-dependent blockade of EA- for insomnia. However, there are concerns regarding the
inducedNREMsleepenhancement(Figures2(a)and2(b)). inappropriate use, dependence and adverse effects of these
Administration of 10μg naloxonazine reduced NREM agents. On the other hand, acupuncture has been used for
sleep from 21.7 ± 2.1% after ketamine + PFS + EA to relievingsleepdisturbancesforthousandsofyearsinChina.
ip
14.9 ± 2.1% (F = 5.100, P = .026, Figures 2(a) and Although acupuncture is efficacious for sleep problems,
(1,190)
2(b)) during the 12-h dark period. Neither naloxone nor especiallyforinsomnia,theunderlyingmechanismswhereby
Evidence-BasedComplementaryandAlternativeMedicine 5
#
50 50
40 40 #
%) 30 30
(
S
M
E
R 20 20
N
∗ ∗
10 10
0 0
∗ 1–4h 5–8h 9–12h
(a) (b)
20
10
15 8
%)
( 6
S 10
M
E
R
4
5
2
∗
∗
0
0
∗ 1–4h 5–8h 9–12h
(c) (d)
∗
100 100
∗ ∗
90
80
#
80
%) 60
(
KE 70
A
W 40
60
20
50
#
40 0
1 2 3 4 5 6 7 8 9 10 11 12 1–4h 5–8h 9–12h
Time(hour)
Shade ipPFS+PFS ipPFS+PFS
ipketamine+PFS ipketamine+PFS
ipketamine+PFS+EA ipketamine+PFS+EA
(e) (f)
Figure1:Theeffectsoflow-frequency(10Hz)stimulationbyEAatAnmian(EX17)acupointsonvigilancestates.The10HzEAenhanced
NREMsleepduringpost-manipulation5–8hduringthesubsequentdarkperiod.∗Representsastatisticallysignificantdifferencebetween
thevaluesobtainedfrom ketamine+PFSand PFS+PFS.Theshadedarearepresentsthevaluesofmean ±SEM.#Depictsastatistically
ip ip
significantdifferencebetweenthevaluesobtainedfrom ketamine+PFS+EAand ketamine+PFS.Theblackhorizontalbaronthex-axis
ip ip
representsthedarkperiodofthe12:12hlight:darkcycle.WAKE,wakefulness.
6 Evidence-BasedComplementaryandAlternativeMedicine
50 25
40 20
∗
∗
%) 30 15
(
S
M
E
R 20 10
N
10 5
0 0
0.1 1 10 0.1 1 10
(μg) (μg)
Naloxone Naloxonazine
(a) (b)
12 4
10
3
8
%)
(
S 6 2
M
E
R
4
1
2
0 0
0.1 1 10 0.1 1 10
(μg) (μg)
Naloxone Naloxonazine
(c) (d)
100
100
∗
∗
90
80
80
%)
( 60
E 70
K
A
W
60 40
50
20
40
0
1 2 3 4 5 6 7 8 9 10 11 12
0.1 1 10 0.1 1 10
Time(hour) (μg) (μg)
ipketamine+PFS+EA Naloxone Naloxonazine
ipketamine+naloxone+EA ipketamine+PFS+EA
ipketamine+naloxonazine+EA ipketamine+naloxone+EA
ipketamine+naloxonazine+EA
(e) (f)
Figure2:TheeffectsofnaloxoneandnaloxonazineonEA-inducedalterationsinsleep.Bothnaloxoneandnaloxonazinedose-dependently
blockedtheEA-inducedenhancementofNREMsleep.∗Representsastatisticallysignificantdifferencebetweenthevaluesobtainedfrom
ketamine+naloxone+EA/ ketamine+naloxonazine+EAand ketamine+PFS+EA.Theblackhorizontalbaronthex-axisrepresent
ip ip ip
thedarkperiodofthe12:12hlight:darkcycle.
Evidence-BasedComplementaryandAlternativeMedicine 7
60 30 ∗ ∗ ∗
50 25
40 20
%)
(
MS 30 15
E
R
N
20 10
10 5
0 0
(a) (b)
30 10
25
8
20
%) 6
(
S 15
M
E 4
R
10
2
5
0 0
(c) (d)
100
100
80
80
%) ∗ ∗ ∗
(
E 60 60
K
A
W
40
40
20
20
1 2 3 4 5 6 7 8 9 10 11 12 0
Time(hour)
Shade ipketamine+PFS ipketamine+PFS
ipketamine+PFS+EA ipketamine+PFS+EA
ipketamine+natrindole+EA ipketamine+natrindole+EA
ipketamine+nor-binaltorphimine+EA ipketamine+nor-binaltorphimine+EA
(e) (f)
Figure 3: The effects of naltrindole and nor-binaltorphimine on EA-induced alterations in sleep. Neither naltrindole nor nor-
binaltorphimine affected EA-induced enhancement of NREM sleep. ∗Represents a statistically significant difference between the values
obtainedfrom ketamine+PFS+EA/ ketamine+natrindole+EA/ ketamine+nor-binaltorphimine+EAand ketamine+PFS.The
ip ip ip ip
shadedarearepresentsthevaluesofmean±SEM.Theblackhorizontalbaronthex-axisrepresentsthedarkperiodofthe12:12hlight:
darkcycle.
8 Evidence-BasedComplementaryandAlternativeMedicine
Table1:Effectsofnaloxoneandnaloxonazineonthealterationsofsleep-wakearchitectureparametersinducedbyEAcofAnmain(EX17)
acupointsinrats.
L:D Numberofboutsa Boutdurationb
Manipulationd Hour cyclee Transitionsc
WAKEf NREMSf REMSf WAKE NREMS REMS
PFS+PFS 1–12 D 3.98±0.31 5.06±0.45 1.58±0.26 18.54±2.20 1.67±0.15 0.67±0.10 26.94±2.31
ip
Ketamine+PFS 1–12 D 3.52±0.36 4.40±0.46 1.20±0.24 24.12±2.97∗ 1.44±0.15 0.59±0.10 25.93±2.62
ip
Ketamine+PFS+
ip 1–12 D 3.43±0.30 4.75±0.46 1.38±0.24 20.57±2.65 2.13±0.20# 0.54±0.09 28.81±2.60
EAc
Ketamine+
ip 1–12 D 3.20±0.44 3.48±0.58 1.21±0.26 29.17±3.53$ 1.15±0.20$ 0.66±0.11 28.22±3.51
naloxone+EAc
Ketamine+
ip 1–12 D 3.19±0.43 4.27±0.65 0.98±0.21$ 26.16±3.30 1.26±0.17$ 0.48±0.10 27.93±3.53
naloxonazine+EAc
Valuesaremeans±SEM.
aNumberofboutsperhour(mean±SEM)foreachvigilancestate;bMean(±SEM)boutduration(min)foreachvigilancestate;cNumberoftransitionsfrom
onebehavioralstatetoanother(mean±SEM)perhour;dExperimentalmanipulation;ePeriodofthelight:darkcycleimmediatelypriortowhichinjections
weregiven:D=darkperiod;fVigilancestates:WAKE,wakefulness;NREMS,non-rapideyemovementsleep;REMS,rapideyemovementssleep;∗Denotes
astatisticallysignificantdifference(P <.05)betweenvaluesobtainedafteradministrationofipPFS+PFSandthoseobtainedafterreceivingipketamine+
PFS;#Depictsastatisticallysignificantdifference(P<.05)betweenvaluesobtainedafteripketamine+PFS+EAcandthoseobtainedafteripketamine+PFS;
$Depictsastatisticallysignificantdifference(P<.05)aftereitherreceivingnaloxoneornaloxonazine.
16 of the ability of EA stimuli at Anmian (EX17) acupoints to
∗ increaseNREMsleep.InordertoperformtheEAstimulation
ht) 14 easily, rats were lightly anesthetized. We found that both
weig 12 ∗ NREM and REM sleep during the first 4h of the dark
ue # # periodweredecreasedafterratsrecoveredfromtheketamine
tiss 10 anesthetic. Ketamine, a cyclohexanone derivative, is used
of
g 8 clinically as a dissociative anesthetic agent both in humans
g/
p and animals. Ketamine is a non-competitive N-methyl- -
hin( 6 aspartate (NMDA) receptor antagonist that blocks catioDn
orp 4 channels[25].Ithasbeendemonstratedthatadministration
nd ofketamineorMK-801,anotherNMDAreceptorantagonist,
-eβ 2 at sub-anesthetic doses produce a robust, dose-dependent
0 increase in the intensity of δ-power of the NREM sleep
Brainstem Hypothalamus Cortex Hippocampus Striatum [26, 27]. Furthermore, the effect of MK-801 by increasing
the metabolic rate in the hippocampus and other limbic
ipketamine+PFS structures stimulates physiological sleep that is similar to
ipketamine+PFS+shamEA thesleepthatfollowssleepdeprivation,indicatingtheneed
ipketamine+PFS+EA ofhomeostaticrecovery[28].Therefore,thesuppressionof
ipketamine+scopolamine+EA NREM and REM sleep after recovery from the ketamine
Figure 4: The effects of scopolamine on the 10Hz EA stimuli- anesthetizationduringthebeginningofthedarkperiodmay
inducedincreaseofβ-endorphin.The10HzEAstimulisignificantly beduetoahomeostaticcompensationtothepreviousanes-
increased the expressions of β-endorphin in the brainstem and thetic state. However, this explanation needs to be further
hippocampus and administration of scopolamine blocked EA- investigated.Ontheotherhand,EAofotheracupointsmay
induced increases of β-endorphin concentration. ∗Represents a exhibitanoppositeeffectonsleeporanesthesia.Forexample,
statisticallysignificantdifferencebetweenthevaluesobtainedfrom EA stimulation of bilateral Neiguan (PC6) shortened the
ketamine+PFS+EAand ketamine+PFS+shamEA.#Depictsa
ip ip post-anesthesia recovery time in cats [29]. However, EA of
statisticallysignificantdifferencebetweenthevaluesobtainedfrom Anmian(EX17)didnotexhibitthiseffectinourexperiment.
ketamine+scopolamine+EAand ketamine+PFS+EA.
ip ip We previously demonstrated that microinjections of
muscarinic receptor antagonist scopolamine into the NTS
andbilaterallesionsofthecaudalNTSblocksthealterations
sleep is improved by acupuncture are poorly understood. in sleep induced by EA stimulation of Anmian acupoints
We,therefore,designedthisstudytodeterminewhetherthe [17], implicating the involvement of cholinergic neurons
opioidergic system of the NTS plays a role in EA-induced in the caudal NTS in this response. Nevertheless, endoge-
alterationsinsleep.OurcurrentresultsdemonstratethatEA nous opiates (β-endorphin, enkepalin, endomorphin and
stimuliofAnmian(EX17)acupointsinanesthetizedratsfor dynorphin) are well known to mediate the analgesic effect
20min enhances NREM sleep during the subsequent dark of EA; low frequency EA stimulation activates μ- and δ-
(active) period. This observation is a further confirmation opioid receptors by the releases of β-endorphin, enkepalin
Evidence-BasedComplementaryandAlternativeMedicine 9
Vagusn. Muscarinic
Ach β-endorphin μ-receptor
receptor
NREM
NTS
Figure5:AhypotheticalmodelbywhichEAAnmian(EX17)mayalterNREMsleep.EAstimuliinthefigurerepresentstheelectricalsign;
Ach:acetylcholine;electricalsign:EAstimuli.
and endomorphin, whereas high-frequency EA stimulation κ-receptors, was then determined by application of specific
activatestheκ-receptorsbyenhancingtheconcentrationsof receptorantagonists.Ourresultsdemonstratethatnaloxon-
dynorphin [30, 31]. It has also been demonstrated that EA azine,aμ-opioidreceptorantagonist,exhibitsasimilardose-
increasesβ-endorphinconcentrationsinthearcuatenucleus dependenteffectasthatofnaloxone,whereasnaltrindole(a
of the hypothalamus via Aβ and Aδ fibers, which in turn δ-opioid receptor antagonist) and nor-binaltorphimine (a
mediatetheanalgesiceffectsofEA[32]. κ-opioid receptor antagonist) have no effect. These results
There are two anatomically distinct β-endorphin path- are in part consistent with the results reported by Reinoso-
ways in the brain; the major pathway originates in the Barbero and de Andres that administration of μ-and δ-
arcuate nucleus and the minor one is in the area of the opioidreceptoragonists,butnotκ-opioidreceptoragonist,
NTS of the caudal medulla [18]. Therefore, it is possibility into the NTS in cats enhances NREM sleep and that REM
thatβ-endorphinconcentrationsmayincreaseinthecaudal sleepisunchangedafterNTSadministrationofthedifferent
NTS after the EA stimulation. Reinoso-Barbero and de opioids [16]. Nevertheless, our results implicate NTS μ-
Andres have shown that microinjection of morphine into opioid receptors as mediators of EA-induced NREM sleep
the NTS provokes a dose-dependent enhancement of the enhancement, which is similar to the effect of μ-opioid
polygraphic and behavioral manifestations of SWS. This receptorsonanalgesiainducedbylow-frequencyEAstimuli.
effectisblockedbynaloxone,suggestingthattheendogenous ThefrequencyofEAweusedinthisstudywas10Hz,which
opioid is involved in controlling electrocortical activity is considered a low-frequency stimulation. Our results also
generated by the NTS [16]. Furthermore, it has been indicatethatconcentrationsofβ-endorphininthebrainstem
documented that vagal afferents activate the β-endorphin and hippocampus increase after EA stimuli, suggesting the
system in the NTS, which in turn mediates responses to involvement of NTS β-endorphin. The enhancement of β-
vagal activation; the effects of acetylcholine on depressive endorphin in the hippocampus may be due to actions of
responses in pulmonary and carotid arteries are mediated multisynaptic relays from the NTS to the hippocampus
by activating the serotonergic and endorphin system of the [34].Enkephalin-andβ-endorphin-containingneuronsare
NTS [33] and the inhibitory effect of vagal afferents on the in the NTS [35]. Therefore, the expression of enkephalin
bradycardiaresponseismediatedbyβ-endorphinergicneu- and endorphin after low-frequency EA stimuli and the
ronsintheNTS[9].Onthebasisofourpreviousresults[17] expressionofdynorphinafterhigh-frequencyEAwouldbeof
and aforementioned evidence, we herein tried to elucidate interestforfurtherinvestigation.Nevertheless,EA-enhanced
whether the effects of EA on sleep regulation is mediated β-endorphin in the NTS is blocked by microinjection of
by the increase of endogenous opiates and the activation muscarinicreceptorantagonistscopolamine,suggestingthat
of opioidergic receptors in the caudal NTS via muscarinic theactivationofendogenousopioidergicsystemismediated
receptors. Application of naloxone, a broad spectrum of bytheNTScholinergicnervoussystem.
opioid receptor antagonist, was used to determine the In summary, our current results demonstrate that EA
involvementofNTSopioidergicreceptorsintheEA-induced stimuli of Anmian (EX17) acupoints enhance NREM sleep
increase of NREM sleep. We found that administration of and this enhancement is blocked by naloxone and nalox-
naloxone directly into the caudal NTS dose-dependently onazine, implicating μ-opioid receptors. Furthermore, the
blocked EA-induced NREM sleep enhancement during the activation of NTS muscrainic receptors after low-frequency
darkperiod,buthadnoeffectontheREMsleep,implicating EA stimuli increases concentrations of β-endorphin, which
the involvement of endogenous opiates in the NTS. A mediates the enhancement of NREM sleep after EA stimuli
potential role for three major opioid receptors, μ-, δ- and of Anmian (EX17) acupoints. A diagram elucidating one
10 Evidence-BasedComplementaryandAlternativeMedicine
hypothetical mechanism by which EA of Anmian (EX17) of the region of the solitary tract,” Archives Italiennes de
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Funding
Electroencephalography and Clinical Neurophysiology, vol. 15,
no.6,pp.969–988,1963.
National Science Council grant NSC95-2320-B-002-098-
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MY2.
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sleep-wakefulnesscyclestatesincats,”Anesthesiology,vol.82,
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in this project. C.-H. Cheng and P.-L. Yi was contributed onsleepactivitiesinrats:theimplicationofthecaudalnucleus
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