Special Issue - Behavioural and Electrophysiological ...
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www.mjms.usm.my © Penerbit Universiti Sains Malaysia, 2015 For permission, please email:[email protected] Abstract Background: Mitragyna speciosa (MS) or ketum is primarily found in Southeast Asia, particularly in northern Malaysia and Thailand. The medicinal value of this plant has attracted significant attention from both herbal medicine practitioners and scientists worldwide. Despite having illegal consumption status, the plant merits study. We conducted a series of experiments to test our hypothesis that ketum impairs both learning and memory in rats. Methods: Ketum leaves were extracted using methanol and standardised for the amou nt of its pure compound, mitragynine. Rats were divided into groups for a passive avoidance task and long-term potentiation (LTP) extracellular recording. In the extracellular recording condition, rats were grouped into control, MS100 (100 mg/kg of ketum extract), MS200 (200 mg/kg of ketum extract), and MS500 (500 mg/kg of ketum extract) groups. An additional group that received morphine was included in the passive avoidance task (10 mg/kg), and there were six animals per group. Rats received daily treatments orally for 28 days for both experiments. Result: Using a passive avoidance task, our data revealed that the rats' memory significantly increased with increasing doses of MS compared to the morphine-treated group. Our findings from LTP recordings showed that LTP was fully blocked by the higher doses of MS. Conclusion: We speculate on the possibility that additional factors were involved in the passive avoidance task because it was an in vivo animal study, while the LTP experiment solely involved brain slices. Keywords: Subchronic exposure, Methanolic extract, Mitrgyna speciosa Introduction Ketum, which is known scientifically as Mitragyna speciosa Korth, has been studied for its potentially positive and negative effects on humans. Ketum may contain mitragynine as a pure compound at up to 66.2% (1) of the total alkaloid content, depending on the region. Takayama (2) calculated that mitragynine comprises approximately 12% of the total alkaloid content in Malaysian species. Ketum has an opiate-like effect and a coca-like stimulant ability that prevents tiredness and increases tolerance for working under high temperature conditions. This plant also acts as a substitute for opium and for weaning addicts off morphine. Pharmacological and phytochemical studies of mitragynine have been conducted to investigate its unique medicinal properties (3–7). Apryani (8) reported Special Issue - Neuroscience Behavioural and Electrophysiological Evidence of Impaired Learning and Memory in Male Sprague Dawley Rats following Subchronic Exposure to Standardised Methanolic Extract of Mitragyna Speciosa Korth Mohd Ulul IlmIe 1 , Sharif Mahsufi mansor 2 , Jafri Malin abdullah 1,3,4 1 Department of Neurosciences, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia 2 Centre for Drug Research, Universiti Sains Malaysia, 11800 Minden, Pulau Pinang, Malaysia 3 Center for Neuroscience Services and Research, Universiti Sains Malaysia, Jalan Hospital Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia 4 Hospital Universiti Sains Malaysia, Jalan Hospital Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia Submitted: 2 Oct 2015 Accepted: 2 Nov 2015 45 Malays J Med Sci. Dec 2015; Special Issue: 45-51
Transcript of Special Issue - Behavioural and Electrophysiological ...
www.mjms.usm.my © Penerbit Universiti Sains Malaysia, 2015For permission, please email:[email protected]
Abstract Background: Mitragyna speciosa (MS) or ketum is primarily found in Southeast Asia,particularly in northernMalaysia and Thailand. Themedicinal value of this plant has attractedsignificant attention from both herbal medicine practitioners and scientists worldwide. Despitehavingillegalconsumptionstatus,theplantmeritsstudy.Weconductedaseriesofexperimentstotestourhypothesisthatketumimpairsbothlearningandmemoryinrats. Methods: Ketum leaves were extracted using methanol and standardised for the amountofitspurecompound,mitragynine.Ratsweredividedintogroupsforapassiveavoidancetaskandlong-termpotentiation(LTP) extracellular recording. In the extracellular recording condition, rats were grouped intocontrol,MS100(100mg/kgofketumextract),MS200(200mg/kgofketumextract),andMS500(500mg/kgofketumextract)groups.Anadditionalgroupthatreceivedmorphinewasincludedinthepassiveavoidancetask(10mg/kg),andthereweresixanimalspergroup.Ratsreceiveddailytreatmentsorallyfor28daysforbothexperiments. Result: Usingapassiveavoidancetask,ourdatarevealedthattherats'memorysignificantlyincreasedwithincreasingdosesofMScomparedtothemorphine-treatedgroup.OurfindingsfromLTPrecordingsshowedthatLTPwasfullyblockedbythehigherdosesofMS. Conclusion: We speculate on the possibility that additional factors were involved in thepassive avoidance task because it was an in vivo animal study,while the LTP experiment solelyinvolvedbrainslices.
Keywords: Subchronic exposure, Methanolic extract, Mitrgyna speciosa
Introduction
Ketum, which is known scientifically asMitragyna speciosa Korth, has been studiedfor its potentially positive and negative effectson humans. Ketum may contain mitragynineas a pure compound at up to 66.2% (1) of thetotal alkaloid content, depending on the region.Takayama (2) calculated that mitragynine
comprisesapproximately12%ofthetotalalkaloidcontent in Malaysian species. Ketum has anopiate-likeeffectandacoca-likestimulantabilitythatpreventstirednessandincreasestoleranceforworkingunderhightemperatureconditions.Thisplantalsoactsasasubstitute foropiumandforweaning addicts off morphine. Pharmacologicaland phytochemical studies of mitragyninehave been conducted to investigate its uniquemedicinalproperties(3–7).Apryani(8)reported
Special Issue - Neuroscience
Behavioural and Electrophysiological Evidence of Impaired Learning and Memory in Male Sprague Dawley Rats following Subchronic Exposure to Standardised Methanolic Extract of Mitragyna Speciosa KorthMohd Ulul IlmIe1, Sharif Mahsufi mansor2, Jafri Malin abdullah1,3,4
1 Department of Neurosciences, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia
2 Centre for Drug Research, Universiti Sains Malaysia, 11800 Minden, Pulau Pinang, Malaysia
3 Center for Neuroscience Services and Research, Universiti Sains Malaysia, Jalan Hospital Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia
4 Hospital Universiti Sains Malaysia, Jalan Hospital Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia
Submitted:2Oct2015Accepted:2Nov2015
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the effects of mitragynine on learning andmemory. Using object location tasks andmotoractivityinanopenfieldtest,theirresultssuggestthat chronically administeredmitragynine couldalter cognitive behavioural function, specificallyworking memory in mice. However, in thepresent study, we focused on the behaviouraland electrophysiological aspects of learningand memory. An experiment in our laboratoryhas demonstrated that acute administration ofmitragynine can induce short-term potentiationintheCA1regionofrathippocampus(9).Asanextensionofthisstudy,herewewereinterestedinstudyingtheinvivoandinvitroeffectsofacrudestandardised methanolic extract of Mitragyna Speciosa (MS). Long-term potentiation (LTP)isacrucialmechanismof learningandmemory.LTP results from coincident activity of pre- andpost-synapticelements,whichcauseafacilitationof chemical transmission that lasts for hours invitro, and persists for weeks or months in vivo(10,11). These results led us to hypothesize thatMS compounds, especially mitragynine, mightimpair the activity of ion channels in neuronalcells, particular those in the dendrites. Thus,the aim of this study was to investigate theeffectsofsubchronicMSexposureonLTPinrathippocampalslicesandonthecognitivefunctionofratsinpassiveavoidanceconditioning.
Materials and Methods
Mitragyna Speciosa Korth Methanolic Extraction and Standardisation The leaves of the plant were collected andthoroughlywashedwithdistilledwatertoremovedirt.Thewetleaveswereweighedandthendriedin an oven at 50 °C for 12 hours. During thistime,the leaveswereperiodicallyturnedovertoprovide uniform drying. The dried leaves weregroundtofinepowderbyamillmachineandthepowderwasweighed.Then,100gofthepowderwas exhaustively Soxhlet extracted in absolutemethanol (100% v/vMeOH) using an extractorand condenser (Ace Soxhlet Extractor 6730,Condenser6740,QuickFit,Staffordshire,UnitedKingdom)for4hoursat60°C.Next,theextractwas concentrated under reduced pressure at 40°Cusingarotaryevaporator.Then,itwasfurtherconcentratedbyallowingittostandovernightinanovenat30°Ctoremoveanytraceofmethanol.Thefinalproductyielded20gofagreenextract,whichwasthenscreenedforthepresenceof thealkaloid mitragynine using gas chromatographymass spectrum (GC-MS). The extract producedwas standardized with reference to the amount
ofmitragyninecontentusingavalidatedGC-MSmethod. Dried extract was stored at 4 °C untilfurtheruse.
Animals Male Sprague-Dawley rats that weighed70–80gofagefourweekswereobtainedfromthebreedingcolonyofAnimalResearchandServiceCentre(ARASC)UniversitiSainsMalaysia(USM).Theywerehousedatadensityoftworatspercageandmaintained at a constant temperature on astandard12h:12hlight/darkcyclewithlightsonat7am.Foodandwaterwereavailableadlibitum.Theexperimentswereconductedaccordingtotheethical norms approved by the Animal Ethics,USM.
Administration Therewerefourgroupsofanimalsinvolvedinthebehaviouraltests,whichwerecontrol,MS100(100mg/kg),MS200 (200mg/kg), andMS500(500 mg/kg). An additional behavioural studyinvolvedagroupthatwastreatedwithmorphine(10 mg/kg). The extracts were administeredto each rat via an oral gavage. The subchronicadministrationofthesedrugsweregivendailyatapproximately10.00amfor28days.
Brain Slice Preparation Transverse hippocampal slices (350 µmthick) were prepared as described by Biessels(12) using a vibrating microtome (HM650V,Microm International, Walldorf, Germany).The slices were maintained in a submergedrecording chamber and perfused (1–2mL/min)with artificial cerebrospinal fluid (aCSF) of thefollowing composition in mmol/l: 124 of NaCl;3.3ofKCl;1.2ofKH2PO4;1.3ofMgSO4;10.0ofglucose;20.0ofNaHCO3;and2.5ofCaCl2andgassedwith95%O2and5%CO2.Allexperimentswerecarriedoutatroomtemperature.Thesliceswere allowed to recover for at least 60minutesincubationafterpreparation.
LTP Recording Field excitatory post-synaptic potentials(fEPSPs)wererecordedinthestratumradiatumof the Schaffer collateral pathway with glassmicro-electrodes filled with the incubationmedium (aCSF) placed at theCA1 region of thehippocampus.Bipolarstainlesssteel stimulationelectrodes,insulatedexceptatthetip,wereplacedon the afferent fibres of the stratum radiatumof the CA3 region of the hippocampus. Beforeeach experiment began, the stimulus intensityto elicit threshold and maximum fEPSPs were
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determined. Next, a stimulus-response relationwas determined and the stimulus intensity wasadjusted to evoke fEPSPs of half maximumamplitude using an amplifier (NPI Electronic,Tamm, Germany) and kept constant thereafter.Stimulation frequencywas0.05Hz.Thefirst 10minutes of every recording served as a baselinevalue.Highfrequencystimulation(HFS)of100-Hz tetanisation was given after the recordedbaselinewasstable.Next, theresponseobtainedwas recorded for approximately 1 hour. TheaverageslopeofthefEPSPatbaselinewassetat100%,andchangesintheslopewereexpressedasachangefrombaseline.Responseswererecordedusing CellWorks version 5.1.1 (NPI Electronic,Tamm,Germany).
Behavioural Assessment: Passive Avoidance ConditioningA passive avoidance task was adapted fromKhajehpour(13)withaslightmodification.Atotalofsixratswereusedinthepassiveavoidance(PA)experiment.Onedayafterthelastadministrationday (day28),eachratunderwentanacquisitiontest in a shuttle box (PACS-30, ColumbusInstruments, Ohio, USA), and the protocol wasset in the software (PASC Shuttle Box V3.13,ColumbusInstruments,Ohio,USA).Theprotocolwasasfollows:a5secondsexplorationduration(thetimearatspentinalightcompartmentbeforetheguillotinedooropened),a2minutesmaximumtrialduration,10mAoftheconditionedstimulus(CS)lightintensity,1.0mAoftheunconditionedstimulus (US) grid intensity, and a 1.5 secondsUSgridduration.TheUSwasgivenimmediatelyaftertheratenteredthedarkcompartment.Thetimetherattooktoenterthedarkcompartmentwas recorded. The experiment was terminatedwhentheratcompletedthe2minutesmaximumtrial duration.For the retention test,whichwasconducted48hoursaftertheacquisitiontest,thesame rat was placed in the light compartment,and 5 seconds later, the guillotine door openedfor 2minuteswithout giving an electrical shockatthedarkcompartment.Thetimetherattooktoenter thedark compartmentwas recorded (stepthroughlatency).
Results
GC-MS Analysis of Standardized Methanolic Mitragyna speciosa Extract The spectrum of the unknown componentswas matched with the spectrum of the knowncomponents stored in the NIST library. TheGC-MS analysis of the active compounds of the
standardised methanolic Mitragyna speciosaextract (MS) showed there were 17 peaks ofcompounds detected in the extract. The mainalkaloid mitragynine was detected at peak 15with a retention time (RT) of 19.51 (Figure 1).Mitragynine accounted for 12.27% of the totalalkaloid andwas themost abundant alkaloid intheplantextract.
Behavioural Studies Thenumberoftrialsintheacquisitionphaseforallgroupswasonly1(resultnotshown),whichindicates that all animals learned the passiveavoidance (PA) task. There were no significantdifferences recorded forall experimental groupsin comparison to the control group for thisphase.Thus,MSinalldoseshasnoeffectonPAacquisition. In the retention phase, there weresignificantdifferencesforallgroupscomparedtothemorphinegroup.ThesefindingsdemonstratedthatMSinalldosesdoesnotimpairthePAtask,as shown bymorphine, andmight increase thememoryoftheanimalsinthehigherdoses(Figure2). Furthermore, the paired t test between theacquisition phase and retention phase for eachgroupshowedstatisticallysignificantdifferences,withtheexceptionofthemorphinegroup.LTP Recording After Subchronic Exposure to MS The LTP data from the brain slices of thesubchronically exposed ratswere obtained fromthe 1 hour recording, which consists of 10 ofminutes baseline responses and 50 minutes ofLTPresponsesthatoccurredpost-highfrequencystimulation. Figure 3 shows the responsesobtainedaftertheHFSforgroupsMS100,MS200,andMS500whencomparedtocontrol.Thefirst
Figure1:Chromatogramofcrudestandardisedmethanolic Mitragyna speciosaextract. Note the elliptical areacoversthepeakofmitragynine.
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10minutes shows the stable baseline responsesrecorded at the CA1 region of the hippocampuswith 0.05 Hz of stimulation. High frequencystimulations consisting of 100 Hz tetani weregiven immediately after the baseline recording.NotethatLTPwasinducedinthecontrolgroup.Recordings from the MS100 group shows thatLTP was lower compared to the control, andprolongedupto40minutes.NoLTPeventswererecordedforbothMS200andMS500.ThepointsrepresentthemeansandtheerrorbarsrepresenttheSEM.TherewasanN=6forthebrainslicesineachexperimentalgroup.Thearrowsindicatethepointofhighfrequencystimulation.
Discussion
The present series of experiments assessedthe role of oral subchronic (28 days) doses ofstandardised methanolic extract of Mitragyna speciosa Korth (MS) on the acquisition andretention of a passive avoidance task, andon the induction and maintenance of long-term potentiation at the CA1 region of therat hippocampus. The results indicate thatsubchronicadministrationofMSathigherdosesdidnotimpairmemory,asassessedinbehaviouralstudies,butinhibitedEPSP-LTPinductionintheCA1regionofthehippocampus.
Figure 2: The effects of different doses ofMSand morphine as a positive control.Thedarkbarsrepresentstep-throughlatency during the acquisition phasewhile the white bars represent theretentionphase,whichwas recorded48 hours later. The error barsrepresent±SEM.*P<0.01and#P<0.001forthecomparisonofallgroupswiththemorphinegroupbyone-wayANOVA.N =6foreachexperimentalgroup.
a
b
c
Figure3:Theeffectsofsubchronically-exposedMS to rats’ brain slices. The first 10minutes shows the stable baselineresponsesrecordedattheCA1regionof the hippocampus with 0.05 Hzof stimulation. High frequencystimulations consisting of 100 Hztetani were given right after thebaselinerecording.NotethatLTPhasbeen induced in the control group.(a)RecordingfromthegroupMS100shows that LTPwas lower comparedto control and prolonged up to 40minutes.(b,c)NoLTPswererecordedfor both MS200 and MS500. Thepoints represent the means, and theerror bars represent the SEM. N =6 brain slices for each experimentalgroup. Arrows indicate the point ofhighfrequencystimulation.
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Behavioural Studies Our experiments showed that subchronicoral administration of 100, 200, and 500 mg/kgofMShavenoeffectonlearninginaPAtaskbecause all groups learned the task. This resultis consistent with an acute study reported bySenik (14). In addition, in the retention phase,the experimental groups and the control grouphad a significantly higher step-through latencycompared to themorphine group. Among all ofthegroups,only theMS500grouphadahigherstep-through latency than the control group,suggesting that MS at higher doses increasesmemoryconsolidation.Apairedttestanalysisoftheacquisitionandretentionphaseshowedthatallgroupsexceptforthemorphinegroupdifferedsignificantly in step-through latency, suggestingthatanimals learnedandbecameconditionedtothetasksintheshuttleboxexperiment. An acute study conducted by Senik (14)for the retention phase showed no significantdifferences among treatment groups. However,the present study contradicted that acute studybecausetheanimalsinourstudymemorisedthepunishment, while the memory in the animalsfromtheacutestudywasimpaired.Furthermore,mitragynine after chronic treatment impairedcognitivefunctioninmice,i.e.,workingmemory(8).We speculate that improvement inmemoryconsolidationintheMS-treatedgroups(especiallyathigherdoses) inthepresentstudywasduetoacombinationoftheperiodofexposureandthevariouscompoundspresentintheextract. Memory impairment was observed inmiceduringastep-downpassivetaskwithsubstantialresidual effects that lasted as long as 24 hoursafterthepre-trainingadministrationofmorphine(15). On the other hand, 30% of the animals inthe morphine-addicted group learned passiveavoidance slower than the control and shamgroup(15). This result is consistent with thepresentstudyinwhichmorphine-treatedanimalslearnedmoreslowlythanothergroups.
Electrophysiological Assessments The electrophysiological results show thatsubchronic oral administration ofMS (200 and500 mg/kg) inhibited LTP induction following100 Hz of tetanus at the Schaffer collateralpathwayofthehippocampalCA1region. LTP is defined as an activity-dependentenduring increase in synaptic activity that isthought to be a cellular correlate of learningand memory. The activation of N-methyl-D-aspartate(NMDA)receptorsinthehippocampusis involved in the inductionofLTPofexcitatory
synaptic transmission. MS is believed to affectthe propagation of EPSP, disrupting integrationby blocking receptors. LTP depends cruciallyon glutamatergic receptor activity, especiallyNMDA and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (17).The connection between synapses is impaireddue to the blockage of the receptors and willthus prevent LTP from being induced (18).Several alkaloids, such as mitragynine(19,20),rhynchophylline(1), andcorynoxineA (21),havebeenreportedtoactascalciumchannelblockers.Inourstudy,wespeculatethattheactionsofthesealkaloidscontributedtotheblockageofLTPattheSchaffercollateralpathwayoftheCA1regionthehippocampus. MS acts as a psychoactive drug similar toopium. Chronic exposure of rats to morphineor heroin induces severe drug tolerance anddependence, andmarkedly reduces the capacityof hippocampal CA1 LTP during the periodof drug withdrawal (22). Eisch (23) reportedthat long-term administration of heroin ormorphine reducedneurogenesis in the adult rathippocampus. Our passive avoidance task data arenot supported by the findings from ourelectrophysiologicalassessments.TheblockingofhippocampalLTPsupposedlyreducesthememoryconsolidation in the shuttle box experiment.Differentpartsandsystemsinthebrainmediatedistinct formsofmemory(24).Thehippocampalandparahippocampalareasofthecerebralcortexconstitute a system that supports declarativeor cognitive memory (25). Other forms of non-declarative memory or memories underlyingother learned behaviours do not crucially relyon thehippocampusandother structuresof themedial temporal lobe, but instead rely on theamygdala, striatum,andcerebellum(26). In thepresent study, electrophysiological assessmentswere performed on brain slices (in vitro) thatpossessed limited neuronal connection, whichmightcontributetothedifferencesinthefindingsfromthebehaviouralperformanceinthepassiveavoidancetask.Wealsospeculatethathormonesplayaroleinmechanismsoflearningandmemoryintheinvivocondition.KorzandFrey(27)foundthat LTP consolidation and memory retrievalwere dependent on β-adrenergic, dopaminergic,andmineralocorticoidreceptoractivities.
Conclusion
In conclusion, MS affects in vivo and invitrolearningandmemorydifferently.Inhibition
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of LTP induction after subchronic exposure toMS was observed in this study, suggesting thatMSmight act on specific receptors in thebrain,particularly receptors in the hippocampus.Furthermore,MSimprovesmemoryconsolidationtohaveapositiveeffectonlearningandmemoryin a passive avoidance task. The standardizedextractofMitragyna speciosamayhavedifferenteffects in vivo compared to the effects in vitro,especially with respect to the functions of thebrain. Additional studies of mitragynine shouldbe performed to further understand the specificmedicinalpropertiesoftheplant.
Acknowledgement
This is to acknowledge a short-term grantfrom the School ofMedical Sciences, UniversitiSainsMalaysia(304/PPSP/6131429).
Funds
None.
Conflicts of Interests
None.
Authors’ Contributions
Conception and design, critical revision of thearticle for important intellectual content, finalapprovalofthearticle,provisionofstudymaterialsorpatients,obtainingoffunding:SMM,JMAAnalysisandinterpretationofthedata,statisticalexpertise:MULAdministrative, technical, or logistic support,draftingofthearticle:SMM,JMA,MUL
Correspondence
JafriMalinAbdullahMD,PhD,FRCS(Ed),FACS,DSCN(Belgium)CenterforNeuroscienceServicesandResearchUniversitiSainsMalaysiaJalanHospitalUniversitiSainsMalaysia16150KubangKerianKotaBharuKelantan,MalaysiaTel:+609-7672083Fax:+609-7672084Email:[email protected]
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