Accepted Article [email protected] E-mail: 303 +(90)262Fax: 03 70 Tel: +(90) 262 303 72 50 41380 Kocaeli-Turkey ofDepartment Pharmacology, FacultyofMedicine Author: *Corresponding 14220 Prague4, Czech Republic Kristina This by is protectedarticle reserved. Allrights copyright. doi: 10.1111/fcp.12386 lead to differences between this version and the throughbeen the copyediting, pagination typesetting, which process, may and proofreading hasThis article been accepted publicationfor andundergone peerfull but review not has c b a Oguz Running title: Article :Original type Article DR. OGUZ MUTLU (Orcid: ID 0000-0003-0952-0742) (AKH/RPCH) family of peptides (AKH/RPCH) familyof on Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i, videnska 1083, National Institute of Mental Health, Topolova 748, 250 67 Klecany, Czech Republic Kocaeli University Medical Faculty, Pharmacology41380, Department,Kocaeli, Turkey Effects of the adipokinetic hormone/red pigment-concentrating hormone Mutlu Holubová

a,b,* Adipokinetic schizophrenia hormone , Tomáš

a,c , Cyril Pálení Oguz MUTLU, M.D., Assoc. Prof. Dr. Prof. Assoc. M.D., MUTLU, Oguz Höschl č ek

a a , Nikola , Aleš , Aleš Stuchlík MK-801-induced schizophrenia models Pinterová Version of Record. Please Version as this cite of Record. article , Kocaeli University

c , Faruk , Faruk

a , Klára Erden Šíchová

b , Karel , Karel

a , Ji Valeš ř í Horá

a,c č

ek

a , Accepted Article Key words: modelschizophrenia ofMK-801. while AKH the inthe in model testinschizophrenia memory improved gating PPI deficits did not modify the MK-801.did notmodifyeffect the of Ani-AKH potentiated caused andPht-HrTH (1mg/kg) byMK-801, the impairment partially task. Lia-AKH (1 mg/kg) significantly potentiated the MK-801-induced PPI disruption, while AAPAin the effectsimpairment memory cognitive MK-801 reversed mg/kg)-induced (0.15 the test. In our study, Ani-AKH (2mg/kg), Lia-AKH (2 mg/kg) (2 Pht-HrTH and mg/kg) MK-801 combination MK-801 of mg/kg), withhormones mg/kg)acutely orthe (0.1 before Pht-HrTH(1 mg/kg), Lia-AKH withrats mg/kg), were (1 treated Ani-AKH (1 Wistar-albino combination of MK-801 thehormones sub-chronically. prepulsewith Inthe test, inhibition MK-801mg/kg), andthemg/kg) Lia-AKH (0.15 mg/kg), Pht-HrTH(2 mg/kg), (2 This by is protectedarticle reserved. Allrights copyright. These pe byan amide. terminally blocked C- and residue pyroglutamate by a blocked N-terminally long, acids 10 amino to 8 from th one of rendering family currently known, this metabolism carbohydrate andprotein control fat, hormone (AKH/RPCH) ofpeptides hypertrehalosemic Adipokinetic peptides [1]. and family pigment-concentrating hormone/red adipokinetic which tothe belongs peptide family, One the groupsof neuropeptide major isthe in insects adipokinetic and hypertrehalosemic INTRODUCTION test Ininhibition(PPI). AAPAthe task, avoidance test (AAPA) and gating sensorimotor MK-801-induced deficit in prepulse the memoryon MK-801-induced in hormone (Pht-HrTH) active the deterioration place allothetic AKH), of theeffects thisin mice. The studyof aim toinvestigate is andexerts neuroprotective effhyperlocomotion AKHshowed antidepressant, that possesses analgesic anxiolytic, and causes effects, controls carbohydrate fat, and protein metabolism insects. Inourprevious in we study, peptides of family The adipokinetic andred (AKH/RPCH) pigment-concentrating hormone ABSTRACT Libellula auripennis Libellula Adipokinetic hormone; schizophrenia; MK-801; rat AKH and (Lia-AKH)

In conclusion, AKHIn insensoriomotorconclusion, no effect had

Longweretreatedwith rats Evans Ani-AKH (2 ptide areproduc hormones ects and increased factorsects and brain neurotrophic e largest family of peptides. All members All peptides. are of family e largest [2]. Approximately 30 different peptidesdifferent are 30 Approximately [2]. Phormia-Terra

Anax imperator Anax ts of neurosecretory ts of hypertrehalosemic hypertrehalosemic AKH (Ani- AKH Accepted Article Thr-Pro-Ser-Trp-NH a sequence of pGlu-Leu-Thr-Phe-Ser-Pro-Asp-Trp-NH sequence a sequence of of sequence dragonfly, the of cardiaca Emperor corpora This by is protectedarticle reserved. Allrights copyright. neuropeptide pGlu-Val-Asn-Phe-Ser-Pro-Ser-Trp-NH cockroaches) Using (inlocusts and heterologous andahomologous bioassay, the a differences Some exist structural variousfunctional AKHs.and insectamong types the of causes hyperlocomotion and exerts neuroprotective effects after chronic injection in mice [9]. (AKH/RPCH) anxiolytic, possesses antidepressant, peptide andfamily analgesic effects, we the study, showedthat our recent [8].cortex In cerebral nucleus periventricular and hypothalamic cord, spinal lumbosacral the of gray dorsal commissure the in present column cell intermediolateral and sacral parasy periventricula and median eminence hypothalamic tolocust antiserum present fibers were adipokineticImmunoreactive intheI [8]. hormone novel peptidergicnervouscentralstudy,systemratby a system was in the identified usingan in th immunoreactivity adipokinetic hormone-like raised antiserum adipokinetic hor locust against neuronal function in the human nervouscentral system study This that an revealed [7]. In previous it studies, was suggested that glutamatergic systemby wasoxytocin shown [6]. andmodulation ofdopaminergic and (PPI) test prepulseinhibition deficits in the oxytocin of effects inthe recent improvement study, Ina [5]. receptors on MK-801-induced superfamily hormone(GnRH)and the vasopressin/oxytocin gonadotropin-releasing of neurons onmotor have effects peptides excitatory energy substrates from fat bodythe many in mediatingof themobilization neuropeptides, metabolic (AKHs)Adipokinetic hormones are cardiaca, neuroethe corpora neurons located in transgenic manipulations of the dAkh gene have demonstrated that AKH plays a role inboth havethe dAkhgene that AKH role plays manipulationstransgenic demonstrated of a using Studies in carbohydrates insects. hemolymph in increase isthe effect hypertrehalosemic while the lipidincreasein the concentration, refers hemolymph the hyperlipemic effect to Libellula auripennis Libellula 2 , while while , Phormia-Terra AKH (Lia-AKH) was determined aspGlu-Val-Asn-Phe- AKH wasdetermined (Lia-AKH) hypertrehalosemic hormone (Pht-HrTH) showed (Pht-HrTH) hormone hypertrehalosemic adipokinetic hormone contributemay tothe Anax imperator Anax mpathetic nucleus. Immunoreactive cells were cells mpathetic Immunoreactive nucleus. mone I can display a mone Icandisplay quantityof considerable insects. Moreover, insects. it is known thatAKH [4]. AKH receptors [4]. are structurally to close ndocrine glandsndocrine brain [3]. attached to the e human cerebrospinal fluid [7]. In another [7]. fluid cerebrospinal e human r nucleus andthe spinalr nucleus corddorsalhorn, 2 was fromisolated theextracts of the 2 [3]. The adipokinetic or The adipokinetic [3]. (Ani-AKH). The amino acid The amino (Ani-AKH). Accepted Article This by is protectedarticle reserved. Allrights copyright. (PPI). test avoidance task (AAPA) and psyc MK-801-induced hormone (Pht-HrTH) memoryon MK-801-induced in activethe deterioration place allothetic AKH), significant behavioural inourpreviouseffects 18]; [9, studies of this to aim studywas the investigate effectsofinsect peptides three have we which found schizophrenia. The modelof in the effects improved shows study AKH to whether important neurotrophic factors in nervous the central syst in depression and AKH improved effects exerts psychosis [17]. ofmodel used be can and apharmacological schizophrenia as PPI and MK-801 disturbs manydiseases in a PPIisdisturbed shown that have Many studies (pulse). strong subsequent stimulus organism to a startling of an reaction in which is phenomenon (PPI) a a neurological successfulmodeling in symptoms ofnegative schizophrenia 15]. Prepulse[14, inhibition of single symptoms injection schizophrenia,positive also a MK-801ofonly was the [13] purposes for experimental psychosis mimic N-methyl-d-aspartatecompetitive (NMDA) rece non- MK-801is frequently used patient’s the (dizocilpine) life. greatlyquality of affect ofin effective Therefore, treatment deficits cognitive life [12]. of schizophrenic patients may Cognitive in is schizophrenia decreases impairment andit common, thequality function and cognitivealso with [11]. dysfunction associated is However, schizophrenia schizophrenia. of the treatment in the primary target were symptoms psychotic positive years, Formany insects [10]. in metabolism in carbohydrate hormone role mostlyplays thehypertrehalosemic a metabolism, while andlipid carbohydrate Libellula auripennis Libellula AKH and (Lia-AKH) nd disorders, especiallynd disorders, inschizophrenia [16]. . Unlike dopaminergic mimic which agonists, . Unlike anxiety by increasing neurogenesis and brain and neurogenesis increasing anxiety by weaker prestimulus (prepulse) inhibits prestimulus inhibits the (prepulse) weaker ptor antagonist inthe animalmodels to em [9, 18];might alsobe therefore, [9, em it hosis model using the prepulse inhibition Phormia-Terra Anax imperator hypertrehalosemic hypertrehalosemic AKH (Ani- AKH Accepted Article This by is protectedarticle reserved. Allrights copyright. Onanimals. 4 the 8 7 and between animalsper groupvaried numberof the and weight, ml/kg body 2 of volume combination orwith before avehicle the AAPA All task. treatments were administered at a in mg/kg) or alone MK-801 (0.15 2mg/kg, a dose hormones of the above-specified at In the AAPA Long task, rats were treated Evans control group. as the 15% DMSOwasused with saline Vehicle consisting of 15% DMSO. with mixed MK-801(Toronto/Canada). was dissolved in saline,AKH while was dissolved saline in United Kingdom. Lia-AKH TRCfrom Pht-HrTH werepurchased Ani-AKH, and maleate,5,10-imine only asMK referred further ((5S,10R)-(+)-5-methyl-10,11- maleate (+)-MK-801 Drugs and treatments (Directive 86/609/EEC). in accordance with Czech and European legislation regarding treatment of laboratory animals were All procedures libitum. ad water receivedfood and animals 7:00). All at cycle (lights on 12/12 light/dark ofa 21°C temperature constant ± and 2°C witha facility conditioned animal air- in an (30×30×40cm) transparent cages plastic housed pairs in were ratsin The to testing, during whichthey were weighing g used for the were PPI 180-250 were rats The acclimatized 7-10daystest. for prior Republic)(Velaz, Czech Wistar rats outbred Male Prague. of Science, Physiology, Academy of Institutethe theaccreditedbreeding obtainedfrom of colony were taskavoidance and Long male Evans rats to four-month-old Three Animals AND METHODS MATERIALS administered administered (i.p.)intraperitoneally for 60 min before the PPI test, while MK-801 was alone or in combination or with a vehicle according paradigm. to this hormonesAll were In the PPI test, the rats treated withwere hormones at 1 mg/kg MK-801 i.p., mg/kg) (0.1 before the AAPA task, while MK-801 was 40 administered min before the test.

th day, the hormones were administered 60 intraperitoneally(i.p.) min weighed twice and handled four times. handled and four weighed twice regularly for 4 days (subchronically) with all all with (subchronically) days 4 for regularly -801) was obtained from Tocris Bioscience,-801) wasobtained Tocris from were used for the active allothetic place dihydro-5H-dibenzo(a, d)-cyclo-hepten- dihydro-5H-dibenzo(a, Accepted Article This by is protectedarticle reserved. Allrights copyright. MK-801-treated ratswere the same us and control wassurface carefully by cleaned adetergent rats. Forsolution between eachof groups, the arena The 0.5 mA. intensity shock of to a respondedappropriately Most freezing.animals mA)was aand 0.7 carefully rat toevoke but preventfor escape reaction rapid each adjusted The [21]. avoidancebehaviorleading rapid to usingprocedure shocks s haspreviously been additional entrances were left counteduntil the than300 Thisthe sector rat for more ms. noratleavedid not sector, additionalshocksms, but the were the 1200 If given every Hz, shock (50 constant-current mild, delivered a the tracking systemthan 500ms(milliseconds), more for shock sector rat enteredthe light-emittinginfrared diode(LED)wasattach an towhich harness, were light latex rats wearing a The in room. located the orienting cues solely distal shocksector relationship bedetermined could tothe of the its spatial by location The rat. the placement of immediately started after arena rotation The USA). Group, Biosignal (iTrack, tracking system computer-based bywas the which defined sector), (shock 60° imperceptible sectora location wasplaced oppositean in the arena to-be-avoided at a rat placedeach trainingdisc.session, beginning thearenaAt the motor an electric underneath of by (1rpm) was wall rotated its togetherThe arena with landmarks. of extra-maze abundance arena was elevated 1mabove floor the of circular(82 cm indiameter), arena byenclosed a30-cm-high transparentwall. The Plexiglas previous in papers detail wasdescribed in arena patient schizophrenic in processing disturbed ofon information depends mode the stimuli, between irrelevant and relevant differentiate successful performance ofthe placeactive allothetic avoidance task, rat has in towhich the believethat intramaze cues). and We stimuli (extramaze spatial discordant subsets of two aconflict solve between to rat requires task Successfulconditions. the performancethe of pathological and their underboth normal efficiently behavior animals toorganize ability of The activeplace allothetic avoidance is aspatialmemory determine the that task test can Active allothetic place avoidance task (AAPA) studies 13,[9, 18, 19, 20].separate A group of was animals usedin each test. dosesthe of administration drugs period and ofdrugs chosen were accordingthe previous to animals per group between varied 12 and 15animal 30administered before min the test in a volume 2of ml/kgweight, body and of number the appropriate shockappropriate (ranging 0.4current between s [13]. The active allothetic place active allotheticavoidance The place s [13]. ed rat’s the between ed shoulders. Whenever the [21]. Briefly, it consists of a smooth metallic metallic smooth a consistsof it Briefly, [21]. a 4×5-m experimental roomcontaining an 4×5-m experimental a hown to be effective and safe for for animals, safe and be effective hown to 0.5 0.4-0.7 0.5 s, mA) and counted an entrance. ed as a reference. Animals areference. in wereed as trained s. Strain of animals used in each test,in each of used animals Strain s. Accepted Article This by is protectedarticle reserved. Allrights copyright. alone trials) six PPI was calculatedtrials. as –follows: [100 (meanprepulse – pulse pulse- trials/mean calculated by percentage the in ASR reduction theinitialsixfrom baseline tothe trials final no stimulus 60-ms.for sixpulse-al Finally, interval inter-stimulus or 120ms) alone: 40 dB; ms/125 prepulse-pulse: (B) 20-ms/ pr were 60trials response Thereafter, (ASR). duration pulse-alone trials were then delivered 75-dB which background white noise was continuously Six 125dB/40 ms-presented. in the chamber 5min to rats startle for acclimatized were The s). 12.27 ITI: 4-20 s(mean of day, acclimatization. werethere Onthe test 72 intotal trials with an (ITI) interval inter-trial for not recorded startle were data The (75dB). noise over white background dB/20 ms) pre-training procedure 5-minute startle test, ratswere acclimatizedTwo the the days the chamber to drug-free, using before a The sound levels were measured usin the average startle amplitudes (AVG) thatwere digitized and used for subsequent analyses. stabilimetersensitivityequivalent cham between Plexiglas stabilimeter (8.7inner in diameter).cm Adynamic calibration systemensured pulse acoustic broadband discrete produced the contai evenlysound-attenuated, enclosure lit startle chambers (SR-LAB,San Diego Instruments, California, USA), each comprisinga was ourpreviously adopted The procedure from (PPI) test inhibition Prepulse session every day, carried out during daylight hours. sessions the active in allotheticplace avoidance lasted task 20 min,hadone andeach rat sector stretching from center ofarenathe itsnorth circumference.to Experimental allothetic dailyfour theactive consecutive sessions shock in avoidance place withthe task, ∗ 100]. The mean ASR was derived from the pulse-alone trials. trials. the derived pulse-alone wasASRfrom 100]. The mean (ISI: mean 70 ms), followed by a 40-ms/125-dB pulse; (C) pulse; a 40-ms/125-dB by followed 70ms), mean (ISI: consisting of 5 presentations of pulse-alone stimuli (115pulse-alone stimuli of of5presentations consisting g a RadioShack soundlevel meter. esented pseudorandomly(A)pulse asfollows: one trials were delivered. Habituation was were delivered.Habituation onetrials ning a high-frequency loudspeaker (which loudspeaker ninga high-frequency bers. A piezoelectric accelerometer detected Apiezoelectric accelerometer bers. 83-dB or91-dBprepulse, 60 a (30, variable published studies Twoventilated published 23]. [22, s and prepulses) mounted 24 cm abovea cm mounted24 s andprepulses) to establish startle to theestablish baseline acoustic Accepted Article This by is protectedarticle reserved. Allrights copyright. p=0.11] ofno significant MK-801 effect [F(1,27)=2.61; revealed two-way2b), ANOVA p=0.0014]. In [F(1,28)=12.53; the AAPA test, wh therewhile wassignificanteffect a andinteraction p=0.006] Ani-AKH of [F(1,28)=8.53; 2a), p=0.11] two-wayANOVA nosignificant revealed ofMK-801effect [F(1,28)=2.65; entries(Fig numberof the evaluated for groupswere whentheAni-AKH In theAAPAtest, group (p<0.01). in the control with Pht-HrTHthat also increased inthe mg/kg+MK-801 group compared 2 while hyperlocomotion, reversing on MK-801-induced the distance total moved effect was mg/kg showed no Pht-HrTH 2 group MK-801 (p<0.05). from statistically different the Ani-AKH+MK-801 was 2 effect. partial had a Lia-AKH while 2mg/kg mg/kg(p<0.05) effectAni-AKH wasreversed 2 by andthis control, in the with(p<0.05) compared that not naivetreatment did affectlocomotion in rats. MK-801 locomotion increased significantly effect of interaction p=0.20]. Bonferroni post hoc[F(1,27)=1.66; test AKH that found whilethere was nosignificant p=0.008] andMK-801 p=0.03] [F(1,27)=7.95; [F(1,27)=4.69; groups (Fig wereevaluated two-way 1c), ANOVA significantrevealed of Pht-HrTH effect and interactionp=0.03] Inthe p=0.03]. AAPA when test, the [F(1,27)=4.98; Pht-HrTH p=0.73] [F(1,27)=0.11; there waswhile a significant effect of MK-801 [F(1,27)=4.76; two-wayevaluated(Fig 1b), reveal ANOVA Inthe p=0.004]. wheninteraction [F(1,28)=9.53; Lia-AKH the AAPA test, groups were eff was asignificant whilethere p=0.46] Ani-AKHof effect significant [F(1,28)=0.54; no revealed ANOVA two-way (Fig 1a), In AAPAthe when test, Ani-AKH the evaluatedgroups were for the totaldistance moved deterioration inthe AAPA test Effects of Ani-AKH, Lia-AKH and Results alpha value was equalbelow to 0.05.or meanasvalues ± SEM. differences The were considered statistically significantwhen the werewere The expressed differences significant data detected. when test hoc post Bonferroni The results of the AAPA andPPI testswere evaluated by two-way ANOVA by followed Evaluation Statistical Pht-HrTH on MK-801-induced memory MK-801-induced on Pht-HrTH ect of MK-801 [F(1,28)=7.29; p=0.01] and p=0.01] [F(1,28)=7.29; MK-801ect of en theLia-AKH were (Figevaluated groups edno significant Lia-AKHeffect of Accepted Article This by is protectedarticle reserved. Allrights copyright. Ani-AKH groups. Pht-HrTH in the significant wasnot effect the while groups, and Lia-AKH treatment significantly decreased the maximum MK-801 hoctest p=0.0062]. post found that Bonferroni [F(1,27)=8.80; of interaction effect p=0.20] [F(1,27)=1.66; and MK-801 while p=0.44] [F(1,27)=0.60; there was significant Pht-HrTH (Fig nosignificant effect two-way evaluated of were revealed ANOVA 4c), groups the when Pht-HrTH AAPA test, the In p=0.14]. MK-801[F(1,27)=2.21; of effect no significant whilethere was p=0.02] interaction [F(1,27)=5.60; and p=0.03] [F(1,27)=4.75; groups two-way (Figwere evaluated of significant ANOVA Lia-AKH 4b), revealed effect significant of MK-801p=0.71]. In effect AAPAthe [F(1,28)=0.13; test, Lia-AKHwhen the while there p=0.0004] wasno interaction p=0.001][F(1,28)=15.96; and [F(1,28)=13.21; Ani-AKH significantof avoidancetwo-way (Fig 4a), effect ANOVArevealed AAPAAni-AKH test,In the the when of groupswereevaluated the time for maximum reversed this (p<0.01) significantly effect. Ani-AKHwhile Lia-AKH 2 and Pht-HrTH (p<0.001), mg/kg 2 mg/kg 2 (p<0.01) mg/kg significantly MK-801 that numberoftest found treatment the increased shocks(p<0.05), hoc Bonferroni post p=0.01]. interaction while of[F(1,27)=7.60; there wassignificant effect p=0.08] andMK-801 p=0.08] effectsignificant of [F(1,27)=3.13; Pht-HrTH[F(1,27)=3.10; no revealed two-wayANOVA evaluated (Fig3c), Pht-HrTHgroups were the when wassignificantwhile interact there of effect p=0.18] p=0.10] MK-801 Lia-AKH [F(1,27)=1.88; and of effect significant [F(1,27)=2.73; no evaluated ANOVA two-way theLia-AKH (Fig revealed when were groups 3b), test,In AAPA p=0.003]. the [F(1,28)=10.29; andinteraction p=0.03] 801 [F(1,28)=4.90; MK- p=0.008], Ani-AKH [F(1,28)=7.89; significant effect of two-wayrevealed ANOVA 3a), when shocks Ani-AKH of (Fig number the evaluated were groupsthe for In theAAPA test, this effect. reversed significantlymg/kg (p<0.01) (p<0.01) and Pht-HrTH2 Lia-AKH (p<0.01), while Ani-AKH the 2 (p<0.001), increased of entries number 2 mg/kg posthocte p=0.007]. Bonferroni [F(1,27)=8.49; while p=0.16] was there of interactionsignificant effect and MK-801[F(1,27)=2.06; two-way2c), ANOVA nosignificant ef revealed IntheAAPA p=0.009]. when test, [F(1,27)=7.93; thePht-HrTHgroupsevaluated (Fig were while there was a significant effect of Lia-AKH [F(1,27)=4.41; p=0.04] and interaction ion [F(1,27)=7.72; p=0.009]. In the AAPA test, AAPAtest, In the p=0.009]. ion [F(1,27)=7.72; st that MK-801significantly found treatment time ofavoidance (p<0.05) Ani-AKH inthe fect of p=0.13]Pht-HrTH [F(1,27)=2.38; Accepted Article This by is protectedarticle reserved. Allrights copyright. MK-801 [F(1,47)=4.59; of effect wassignificant there while p=0.95] [F(1,47)=0.002; was alsono p=0.13] significant [F(1,47)=2.29; and of effect interaction Pht-HrTH There onthe ASR p=0.65] data. [F(1,47)=0.20; andinteraction p=0.05] 801 [F(1,47)=3.74; MK- p=0.89], nosignificant effectTwo-way ofPht-HrTH[F(1,47)=0.01; ANOVA revealed also a significant effect betweentheMK-801-alone and combination groups (p<0.001). was the vehicle There compared with that group. in control combination groups (p<0.001) found that %PPI was decreasedsignificantly p<0.0001][F(1,4 andinteraction [F(1,47)=51.80; MK-801 p=0.0002], [F(1,47)=15.99; Lia-AKHof effect significant revealed way ANOVA two- (Fig the evaluated when PPI 6), groups Lia-AKH were the In test, between groups. onthe habituation data p=0.01] although posthoctestsno showed significant difference MK-801 [F(1,47)=6.38; of effect wassignificant there while p=0.95] [F(1,47)=0.003; and interaction p=0.75] of [F(1,47)=0.09; Lia-AKH significant effect also no was There onthe ASR p=0.84] data. [F(1,47)=0.03; andinteraction p=0.09] 801 [F(1,47)=2.87; p=0.33], MK- Two-way Lia-AKH effect nosignificant ANOVA of revealed [F(1,47)=0.96; (p<0.001) comparedthat with in the vehicle group. control MK-801and1mg/kg 0.1-alone combination group groups group(p<0.05), (p<0.001) Bonferroni posthoctest %PPI that found wassignificantly decreased Ani-AKH inthe p=0.34]. of interaction[F(1,47)=0.90; effect significant while wasno there p<0.0001] MK-801 [F(1,47)=20.67; and p=0.01] [F(1,47)=5.92; Ani-AKH of significant effect revealed PPIwhen two-way(Fig evaluatedgroupsthe theANOVA Ani-AKHwere groups. test, 5), In tests althoughdata posthoc habituation on the p=0.03]theresignificant ofeffect MK-801 was while p=0.50] [F(1,47)=4.59; [F(1,47)=0.44; nowas also significant effectAni-AK of There onthe ASR p=0.62] data. [F(1,47)=0.24; andinteraction p=0.07] 801 [F(1,47)=3.20; MK- p=0.61], Ani-AKH nosignificant of effect [F(1,47)=0.26; ANOVATwo-way revealed in the PPI deficits test Effects of Ani-AKH, Lia-AKH and Pht-HrT MK-801-induced decrease in the maximum timeofavoidance. themg/kg significantly reversed Lia-AKH (p<0.05) 2 and 2 (p<0.001), Pht-HrTH 2(p<0.01) H gating MK-801-induced sensorimotor on H [F(1,47)=1.45; p=0.23] [F(1,47)=1.45; H and interaction showed no significant difference between the showed nosignificant in the MK-801 0.1-alone (p<0.01) and theMK-801 (p<0.01) in 0.1-alone 7)=4.21; p=0.04]. Bonferroni posthoctest p=0.04]. 7)=4.21; Accepted Article This by is protectedarticle reserved. Allrights copyright. memory andsensorimotor gating can also be ininvestigatedstudies. future AKH administration of on the chronic effects in The naive animals. of memory deterioration Thisstudy supports [18]. that AKH/RPCH the fa our recentpassive avoidance in in acute the administration memory deterioration testafter no AKH Similarly, in caused animals. naive observedsubchronicallyno memory and deficit the drugs weadministered IntheAAPA test, thePPItest. gating in sensorimotor disturbed AAPAand test hypermotility in the and deterioration memoryMK-801 caused in ourstudy, studies, previous to Similar 23, 24]. [13, impairment memory MK-801 induced on had improving sertindoleeffects olanzapine, clozapine antipsychoticsstudies and like and known thatMK-801 iswell disturbes memo It 23]. had while generationschizophrenia new antipsychotics improving [13, models [13], effects induced inMK-801 oncognition effects improving known have were notto haloperidol like antipsychotics Classical [13]. and cognitivedeficits processing sensorimotor and stereotypy), hyperlocomotion(e.g.,symptoms positive-like induce modelsisknownto animal MK-801 in induced1mg/kgdeficit. sensorimotor gating none of the hormonesthree inducedalone deterioration memory only while Ani-AKH Finally, no effect. had while Ani-AKHand Pht-HrTH potentialization, 801, partial showed In Lia-AKH deficitthe PPI the bytest, AKH induced butnot Pht-HrTH. MK- by potentiated increasethe MK-801-induced in locomotion was byLia- byreversed andpartially Ani-AKH the MK-801-induced effects cognitive impairment in memory AAPA the task. Furthermore, reverse hormonescould with all subchronic treatment study wasthat findingmain our of The DISCUSSION that inthe vehicle group.control (p<0.01) 0.1-alone MK-801 the decreased in posthocBonferroni p=0.0003]. test [F(1,47)=15.18; found %PPIwassignificantly that while there p=0.24] interaction [F(1,47)=1.39; was significant of MK-801 effect p=0.19] and Pht-HrTH[F(1,47)=1.69; no significant effectway of ANOVArevealed two- (Fig 7), evaluated groupswere Pht-HrTH whenthe test, PPI groups. Inthe the between onthe habituation data p=0.03] although posthoctestsno showed significant difference and combination group (p<0.01) compared with compared groupcombination (p<0.01) and mily of peptidesmily donot of psychosiscause or ry similar to schizophrenia in ourpreviousto schizophrenia in ry similar

Accepted Article This by is protectedarticle reserved. Allrights copyright. resulting the C, and phospholipase and receptor activate a Gq-protein-coupled AKHs to bind byendogenous AKH, lipids of and carbohydrates metabolism carbohydrates. Duringthe pa usesimilar They [31]. glucagon inhumans inas Adipokinetichormoneinknown insectsof is analog humans the mobilization [30]. cause lipidhormone family these theadipokinetic also shownthatof peptides insect carbohydrates and lipids and/or theutilization of su of mobilization onthe effects direct have hormones These insects. metabolism in protein and carbohydrate fat, hypertrehalosemic peptidescontrol Adipokinetic and significanteffect of all three hormoneson all parameters of cognition in the AAPAtest. locomotion and of performance cognitive the theanimals in AAPA test although was there cancausesome nonspesific effectsto the cognitive task. Anxiolytic effect AKH of can affect effect.situation This this while Ani-AKH AAPAmovedLia-AKHreversed inthe test and actively zone can go the orpassively. outof shock the zonerat canenter either in two ways, the AAPA test, In the AAPA inthe test. of animals memory the performance have increased theThus, anxiolytic effect of AKH andmodul NMDA18]. antagonists such as anxiolyticantidepressant and of effects AKH affect cansuchanxiety, nonspecific factors, as some the test, AAPA in Additionally, 28]. [26, in schizophrenia neurotrophic factors AKHreversing theenhancementof deterioration onmemory effect ofthedecreased canbe enhancement effect of these drugs 26, [25, levels,CREB which is thought tobe responsible for the pro-cognitive and mood while antidepressant and antipsychotic therapyincreased diminishedlevelsthe of BDNFand theBDNFschizophrenia, weredecreased andCREBlevels hippocampus in of the patients shown that, Itwas indepression andmice twoweeks after of administration. intraperitoneal and ( bindingc-AMP element response protein the of AKH that administration chronic increased significantly increased the impairedof MK-801. In weeffect study ourprevious showed [9], although AKH didnotreverse MK-801-indu MK-801-induced cognition deficits found thatAAPA allthe hormonesreversed in the test, and MK-801801-induced memory deterioration We obtained contrasting whenwe results theeffects evaluated of threehormoneson MK- MK-801 known possess arealso to [29]. anxiolytic effects In our study, MK-801 increased total total MK-801distance increased our study, In both after acute and [9, chronic administration acute and after both 27]. In our study, one explanation for the explanationfor 27]. In ourstudy, one passively or actively, and in a similar way, it asimilar and actively, in or passively the results. In ourrecent study, we showed ating with MK-801 on effect anxiety could CREB) levels CREB) levels hippocampusin ofthe naive thways for the mobilization of lipids and lipids of mobilization the thways for -induced sensorimotor gating deficits. We gating deficits. sensorimotor -induced brain-derived neurotrophic factor (BDNF) factor neurotrophic brain-derived ced PPI disruption, although Lia-AKH although PPIdisruption, ced ch substrates by flight muscles [3]. It was It [3]. musclesch substratesby flight Accepted Article This by is protectedarticle reserved. Allrights copyright. and quetiapine olanzapine, risperidone, antipsychoticsAtypical clozapine, such as onnegative schizophrenia buthave no effect symp Classical antipsychotics such known as haloperidol toimproveare the positive symptoms of for sensorimotorthe increaseMK-801-induced in gating deficits. alterations indop model while a schizophrenia on memory in improved effects the in cholinergic causemaythat alterations AKH by activity Alzheimer’s in effects improved shows the brain in activity increasedcholinergic that known AKH. is It activityserotoninergic decrease may increase in cholinergic activity may AKH, increase while a decrease indopaminergic and study, it Accordingcan hormone behypothesized tothis lipid bythat [34]. adipokinetic an blockersmethysergidereceptor dopaminergicpropranolol, receptorchlorpromazine serotonergicand flupenthixol, blockers alpha adrenergic receptor blocker phentolamine, beta adrenergic receptor blocker Reserpin e the reversed adipokinetic hormone. byincreasing study, anticholinesterases lipid hemolymph this increased In insects [34]. of anticholinesterases, is reducedby nervous ofsystem monoamines the content depleting in the In itwasa previous study, which shown that adipokinetic berelease, hormone can by induced observed in ourstudy. and synerg canexplainsome this receptors, NMDA deactivationmayof peptidesof the AKH/RPCH effectcause mobilization family incultured NMDARdesensitization increased NMDAR cholesterol profoundly responsesresulting incell depletion diminished and itandchronicwasthatstudy,activity acute pretreatments shown In that wasshown[33]. of modulation NMDAR membrane study, roleof recent the lipids physiological In in the a studies. in further the role in central effects ofAKH experime in diacylglycerols orfree fatty acids Thes [32]. extracellular Ca the cyclic with together AMP, messengers intra- andcoupled receptor. The resulting sustained flight, adenylate is activatedcyclase after the AKHs to bind Gsprotein- their inositol trisphosphate releases Ca 2+ , , activate a triacylglycerol lipase, which results in the of production 1,2

and gramine all reversed the increased effect in hemolymph effect theincreased gramine reversed all and 2+ from internal stores. In insects, which use lipids for uselipids which Ininsects, internal stores. from istic effectsistic of AKH with MK-801, whichwe e second messenger pathways may a alsoplay ntal investigatedmodels andshould animal be amine/serotonin receptors receptors beresponsiblemay amine/serotonin ffect of AKH by depleting monoamines. The AKH The monoamines. of depleting ffect by disease [35]. In our study, we can speculate wecan our study, In disease [35]. rat cerebellar granule cells. The lipid The granule cells. rat cerebellar toms such such as toms 37].cognition deficits [36, Accepted Article This by is protectedarticle reserved. Allrights copyright. studies [6]. distinct effects has more oxytocin However, 18]. to oxy to becomparable seem effects central its thatto of oxytocin, and AKH structure asimilar [41]. has nervousfor oxytocin central system oxytocinintranasalhas of administration been proposed possibleas routeof deliverya to the mayand itsdeficit pathophysiology ininvolved [40]. The be inthe depression humans of produces antidepressant-lik also Oxytocin [38]. oxytocin hasadministered beenreported to reduce possiblyfear, by inhibiting the amygdala cognitive and which function behavior, are in schizophrenia disturbed [6]. normal theregulationplays of rolein that peptide animportant acid nine-amino Oxytocinis a studies. be for this further can important finding also not onmemory, positive the but symptoms on Sincemanydrugsmodel. improvinghave effects antipsychotic schizophrenia the in memory AKH of on improvingthe effects showswhich AAPAtest related with positive symptoms of schizophrenia butimproving wefound in the effects is which test of PPI AKH in the effect findcouldn’t we ourstudy, Soin schizophrenia. of symptoms positive morely includes which gating deficits sensoriomotor with related is test weinvestigatedwhich effects onmemory dete ta with different related were PPI tests AAPA and Wistar-albino ratsfor the as PPItest inprevious studies [13, 19, 20]. the AAPA test since they were used more frequently in the cognition tests while we used we strainsdrugs used different of inanimals were used Long-Ewans rats for and ourstudy. deficits.Difference tests betweenthe two canbealsosourced from the different dosesof the gating andsensorimotor cognition, locomotion playingvarious in brainparts roles different results can duebe also to thedifferent structures and ofthe functions insect hormones and demonstrated on gating sensorimotor deficits 801-induced hyperlocomotion in the AAPA however, test; no improvement were effects act as a orclassical an atypical drugantipsychotic of because the tendency toimprove MK- disturbed memory 37]. results emotion The and of ourstudy[36, showed AKH that didnot demonstrated have aripiprazole impr also ovedeffects onnegative as symptomssuch rioration in theschizophrenia modelwhilePPI rioration in the PPI test. The controversy between our our between controversy The test. PPI the in e effects in animal models of depression [39], modelsof effects inanimal e tocin in this study and in previous studies [9,in previousstudies studyin this and tocin than AKH in schizophrenia models in recent models AKHin schizophrenia than sks. APPA test is related with cognition in in cognition related with is APPAtest sks.

Nasally Accepted Article This by is protectedarticle reserved. Allrights copyright. Education, Youth and Sports of the Czech Republic under the NPU I program undergrantHealth”, numberLO1611, a with support from financial Ministry the of Mental of National Institute the “Sustainability for project the by and Czech Republic the Agency Grant the of This work P304/12/G069 andP304/14/20613S of was supportedfrom ACKNOWLEDGEMENTS MK-801 the PPItest. in Ani-AKH andLia-AKH because didit notalter sensorimotor inducedthe gating by deficits inschizophrenia, whiledysfunction Pht-HrTH tohave seems a superiorto those effect of improve theAKH/RPCH peptides could cognitive that family This study documents CONCLUSION after two weeks of intraperitoneal injection [9]. highest dentate hippocampus gyrus neurogenesis mice andleast inthe neurodegeneration of causedhighesneurodegeneration the least and This waseffect. alsoeffect with line our in of MK-801,the impairing effect while Ani-AK PPI test, Inthe previous[18]. study findings hyperlocomotion,Ani-AKH withourcorresponded a that MK-801-induced finding decreased the we and drugs IntheAAPAadministered administered [18]. subchronically test, acutely locomotion while [9], Ani-AKH at locomotion the 4-mg/kgdiminished when dose found that when these hormoneswere administered theychronically, all increased the distance weincreased studies, combined when total moved recent withMK-801. Inour causedhyperlocomotion while Lia-AKH by hadapartial Pht-HrTH effect, while MK-801 inthe For AAPAAni-AKH of insect example, hormones. types test, of different reversed weIn thisstudy, some behavioral also found that could differ effects the afteradministration previous study in which we found that Pht-HrTH we Pht-HrTH foundwhich that studyprevious in we found that Lia-AKH significantlywe foundthat enhanced H had a partial effect and Pht-HrTH had no and hadno apartial Pht-HrTH effect H had t neurogenesis, while Lia-AKH while tneurogenesis, the caused Accepted Article This by is protectedarticle reserved. Allrights copyright. 6. from corpora the cardiacaEmperor ofthe [10] Gade G., KellnerJanssens M.P., R. A novel peptide in the AKH/RPCH isolatedfamily Fundam. Clin. Pharmacol. (2016) behavior, BDNF andCREB expression in th ofchronicEffects of administration adipokinetic and hypertrehalosemic hormoneanimal on Mutlu[9] O., Gumuslu UlakS., Kokturk E., AkarG., F.,F.,ErdenKaya H., TanyeriP. (1985) revealshormone a novel systempeptidergic in rat the nervouscentral system. Res.Brain Sasek[8] C.A., Schueler Herman P.A., R.P. EldeW.S., Anantiserum to locust adipokinetic (1989) immunoreactivity the in human cerebrospinal fluid.Biochem. Biophys. Res. Commun. Identification andof characterization adipokinetic migratoria)-like hormone (Locusta Suzuki SatoH., [7] TsuchiyaS., N., SuzukiT., Ishihara Muramatsu H., ShimodaS. Y., gating. (1999) Psychopharmacology (Berl). [6] Feifel D., Reza T. Oxytocin modulates psychotomimetic-induced deficits in sensorimotor Endocrinol. (2011) [5] RochG.J., BusbySherwoodN.M. E.R.,Evolution ofGnRH: diving deeper. Gen. Comp. melanogaster. Genetics (2004) byaffected genetic of manipulations thegenein adipokinetic hormone-encoding Drosophila Lee[4] ParkG., J.H. Hemolymph homeostasis sugar inducedand starvation hyperactivity Chem. Org.Naturst. (1997) GädeG.[3] Theexplosionof structuralinsect information on neuropeptides. Fortschrm. (1992) [2] Gade G. The hormonal integration of insect flight metabolism. Zool. Jahrb. Physiol. (2008) 44–45.of Entomology [1] Gade G., Marco H.G. Adipokinetic and hypertrehalosemic neurohormones. Encyclopedia REFERENCES 96 343 163 211. 172-175. 534-540. 171 1-16. 71 167 1-128. 30 311-323. 4-13. dragonfly, Anaxdragonfly, imperator. Peptides.(1994) 141 e hippocampus and neurogenesis in mice. 93-98. 15 1- Accepted Article This by is protectedarticle reserved. Allrights copyright. cues byrotationof the arena. Proc. Natl. Acad.USA. Sci.(2001) impairs avoidance ofa stableroomdefined pla [21] Cimadevilla J.M., Wesierska M., Fenton A.A., Bures J.Inactivating one hippocampus Neuropharmacology (2007) low highanda dose of1-Aagonist serotonin on behavior inducedby MK-801. [20] Bubenikova-Valesova V., Votova M., Paleni Behav. (2013) schizophrenia induced by acute MK-801: An effect of pretraining. Pharmacol. Biochem. A. I.,memory working impairedFajnerova Visuospatial Stuchlik in is modelanimal an of ZemanovaLobellova A.,[19] Stankova A., V., SvobodaVales J.,Vlcek K.,K., Kubik S., Locomotion and Memory Mice. in Chin. J.Physiology (2017) Effects of Acute Administration of Adipokinetic Hormone on Depression, Anxiety, Pain, [18] MutluO., UlakG., Akar F.,Erden F., CelikyurtI. K., Bektas E., TanyeriP., KayaH. (1999) Neuropsychopharmacology. to hypofunctionhypothesisreceptor dopamine the ofschizophrenia. Jentsch[17] J.D., Roth R.H. Theneuropsychopharmacology phencyclidine:of NMDA from review. Psychopharmacology. (2001) of prepulse inhibition models ofsensorimotor gatingdeficitsA in schizophrenia: decade in [16] Geyer M.A., Krebs-Thomson K., Braff D.L., Swerdlow N.R. Pharmacological studies Behav. Brain Res. (2003) [15] RiedelG., Platt B., Micheau J. Glutamatereceptor function inlearningandmemory. processes.Targets Drug Curr. (2001) [14] Castellano C.,Cestari V., Ciamei A.NMDAreceptors and learning andmemory avodance task. Proc. Natl. Acad. Sci. USA. (2008) ritanserin but nothaloperidol block effect of [13] Bubenikova-ValesovaA., V., Stuchlik Svoboda Bures J., J., Vales K.Risperidone and (1999) Bull. Schizophr. Addington[12] J., Addington D. Neurocognitive social and functioningschizophrenia.in 119-136. outcome in schizophrenia:are we measuring [11] GreenM.F., KernR.S., Braff D.I.,Mint 106 117-123. 25 140 173-182. 52 1-47. 1071-1078. 1071-1078. 20 2 156 201-225. 273-283. 117-154. dizocilpine on the active allothetic place z J. Neurocognitive deficits and functional the “right stuff”. Schizophr. Bull. (2000) ce during dissociationof arenacues room from cek T., Horacek J. The J. Horacek T., oppositecek effect ofa 105 1061-1066. 60 106-113. 106-113. 98 3531-3536. 26

Accepted Article This by is protectedarticle reserved. Allrights copyright. family. General Comparative Endocrinology (2003) GadeG.,[32] Auerswald Mode L. of ofaction 3103-3110. Drosophila modulates both lipidand carbohydrate homeostasis. J.Exp. Biol. (2008) [31] Bharucha K. N.,P.,Zipursky Tarr L.S. glucagon-like A pathway in endocrine inhumans.lipid mobilization USPatent(2005) US 6852693B2. [30] Schacter B., Wilmington D.E., Schacter L.P. Compositions and methods promotingfor 101–107. antagonists in anelevated plus-maze model ofanxietyinmice. Eur. J. Pharmacol. (1995) [29] Wiley J.L., Cristello A.F., Balster R.L. Effects of site-selective NMDA receptor neurotrophic factor expression in ra Quetiapine attenuates immobilizationthe stress-induced decrease ofbrain-derived Xua[28] H.,Qinga H.,Lub W., KeeganaD., RichardsoncJ.S., Chlan-Fourneya J., LiaX.M. (2006) Psychiatry Neuropsychopharmacol. Biol. neurotrophicderived factor (BDNF)changes in the serumof depressedProg. women. [27] Aydemir C., Yalcin AksarayE.S., S., Kisa C., Yildirim S.G., Uzbay T., Goka E. Brain- Res. (1999) neurotrophic factor results in impairment of spa Mu [26] YuoZ.B., LiW.P.,J.S., Zhouc X.F. Deprivation of endogenous brain-derived onEffects transcription gene hippocampus.the in Rev. Neurosci. (2005) Alfonso[25] J., FraschA.C., Flugge G. Chr mice.Biochem. Behav. Pharmacol.MK-801-treated (2011) sertindole and clozapine on learning and memory in the Morris water maze test in naive and Mutlu[24] O., UlakG., I.K.,Celikyurt Akar Yıldız Effects F.,ErdenF. of olanzapine, Biochem. Behav. (2005) risperidone, clozapine andolanzapine onMK- [23] BubenikovaVotava V.,M., Horacek Dockery T., J., C. Palenicek The ofzotepine, effect Prog. Neuro-Psychopharmacol. Psychiatry Biol. (2016) Pharmacokinetics,aminoindane (MDAI): behavi 5,6-methylenedioxy-2- of toxicity Emerging R.R. Horsley F., K., Tyls Vales M., Cegan M., [22] Palenicek T., Lhotkova E., Zidkova M., Balikova M., Kuchar M., Himl M., Miksatkova 835 259-265. 80 591-596. t hippocampus. Neurosci. Lett. (2002) onic stress, depression and antidepressants: 801 disrupted801 sensorimotor gating. Pharmacol. neuropeptides neuropeptides thefrom adipokinetichormone tiallearning and in memoryBrain adultrats. our, thermoregulation and LDL50 in rats. rats. in LDL50 and thermoregulation our, 30 1256-1260. 132 69 10–20. 49-59. 49-59. 98 398-404. 16 321 43-56. 65–68. 211

294

Accepted Article This by is protectedarticle reserved. Allrights copyright. (2014) OxytocinConcentration Changes following intranasal delivery Macaque. in PLoS One. MonteO.D.,[41] Turchi Cummins Noble J.,A., P.L., Averbeck B.B.CSF and Blood understanding depression". Neurosci. Biobehav. Reviews.(2014) McQuaidO.A.,[40] R.J., McInnis A., Abizaid Anisman H. room "Making oxytocin for in J.(2012) disorders". Sci.for mental Physiolog. Matsuzaki[39] MatsushitaH., M., Tomizawa K., H.Matsui therapeutic a "Oxytocin: target and fearin humans". J.Neurosci. (2005) B.,Gallhofer A. Meyer-Lindenberg Oxytocin modulatesneural circuitry forsocial cognition KirschEsslinger[38] P., C., Q.,Chen Mier Lis D., GruppeS., Siddhanti S., Mattay H., V.S., antidepressants imipramine andparoxetine.Behav. (2003) Pharmacol. antipsychotic haloperidol, the atypical antipsychotics clozapine and olanzapine, and the latenteffects in inhibition the betweenand differentiatesforced swim test typical the I.,[37] Weiner Schiller I., Gaisler-Salomon D., A., Green Joel D. A of drugcomparison effects onneurogenesis. Psychiatr Danub. (2012) NandraK.S., Agius[36] TheM. betw differences 1703–1718. ofprogression Alzheimer's disease: therapeuti MufsonE.J.,[35] Perez Counts Ginsberg S.E., S.E., Cholinergic system S.D. theduring inhormonelocust the schistocerca gregaria. Exp. J. Biol. (1976) Samaranayaka B.[34] Possibleinvolvementthein ofmonoaminesofrelease adipokinetic NMDAprobability andPhysiol.(2015) J. desensitization of receptors. KrusekJ., BalikT., A., Smejkalova HorakM., VyklickyCholesterol L. modulates open KorinekM.,[33] Vyklicky V., Borovska J., Lichnerova K., Kaniakova M., KrausovaB., 9 e103677. 25 11489–11493. c implications. Expert Neurother.Rev. (2008) 62 een typicaleen andatypical antipsychotics: the 24 441–444. 95-99. 65 45 415-425. 305–322. 14 593 215-222. 2279–2293. 8

Accepted Article (AAPA) in Long-Ewans rats. The data are indicated as the means ± standard error of ± means of the the as error standard are data indicated The rats. Long-Ewans (AAPA) in 4 (n=8), for days place Pht-HrTH2+MK-801 inthe active(n=7) allothetic avoidance test 2+MK-801Lia 2+MK-801Ani (n=8), mg/kg)(0.15 MK-801 (n=8), (n=8), HrTH)(2 mg/kg) (n=8), Lia-AKH ( Lia-AKH (n=8), ( Ani-AKH (n=8), vehicle administering (i.p.) Figure 4 compared with the group). MK-801-alone mean ± means of the the as error standard are data indicated The rats. Long-Ewans (AAPA) in 4 for (n=8), placedays Pht-HrTH2+MK-801 inthe active(n=7) allothetic avoidance test 2+MK-801Lia 2+MK-801Ani (n=8), mg/kg)(0.15 MK-801 (n=8), (n=8), HrTH)(2 mg/kg) the mean of error standard place avoidance test (AAPA) in Long-Ewans rats. The data are indicated theas means ± allothetic active the in days 4 for (n=7) 2+MK-801 Pht-HrTH (n=8), 2+MK-801 Lia (n=8), ( (n=8),Lia-AKH vehicle (i.p.) ( Figure 2 with the MK-801-alone group). compared group;#p<0.05 control compared the (*p<0.05, **p<0.01, ***p<0.001 with mean ± means of the the as error standard are data indicated The rats. Long-Ewans (AAPA) in 4 for (n=8), placedays Pht-HrTH2+MK-801 inthe active(n=7) allothetic avoidance test 2+MK-801Lia 2+MK-801Ani (n=8), mg/kg)(0.15 MK-801 (n=8), (n=8), HrTH)(2 mg/kg) This by is protectedarticle reserved. Allrights copyright. ( Lia-AKH (n=8), (n=8),( Ani-AKH vehicle administering Figure 3 ###p<0.001 compared with the MK-801-alone group). ( Lia-AKH (n=8), (n=8),( Ani-AKH vehicle administering Figure 1 LEGENDSFIGURE Phormia-Terrae

(*p<0.05, **p<0.01 compared with the control group; ##p<0.01, ###p<0.001 control group; ##p<0.01, the with **p<0.01 compared (*p<0.05, illustrates the total distance moved (m) data obtained by intraperitoneally (i.p.) obtainedintraperitoneally by (m) data moved distance thetotal illustrates illustrates the number of shocks obtaineddata by (i.p.) intraperitoneally illustrates the maximum time of (s)dataobtainedavoidance by intraperitoneally illustrates the of number entries data obtained by intraperitoneally administering HrTH) (2 mg/kg) (n=8), MK-801 (0.15 mg/kg) (n=8), Ani2+MK-801 mg/kg) MK-801 (n=8), (0.15 (2mg/kg) (n=8), HrTH) Libellula Auripennis Libellula Libellula Auripennis Libellula Auripennis Libellula

(**p<0.01 compared with the control group; ##p<0.01, group;##p<0.01, control with the compared (**p<0.01 Libellula Auripennis Libellula AKH) (2 mg/kg) (n=7), Pht-HrTH ( Pht-HrTH AKH)(2 (n=7), mg/kg) AKH) (2 mg/kg) (n=7), Pht-HrTH ( Pht-HrTH AKH)(2 (n=7), mg/kg) AKH) (2 mg/kg) (n=7), Pht-HrTH ( Pht-HrTH AKH)(2 (n=7), mg/kg) Anax Imperator Mauricianus Anax Anax Imperator Mauricianus Anax Anax Imperator Mauricianus Anax

AKH) (2 mg/kg) (n=7), Pht-HrTH AKH) Pht-HrTH (n=7), (2 mg/kg) AKH) (2 mg/kg) (2 AKH) mg/kg) AKH) (2 mg/kg) (2 AKH) mg/kg) AKH) (2 mg/kg) (2 mg/kg) AKH) Phormia-Terrae Phormia-Terrae Phormia-Terrae

Accepted Article This by is protectedarticle reserved. Allrights copyright. (**p<0.01 compared to the control group). test (PPI) in rats.Wistar The indicated data are means the as ± standard error meanof the mg/kg) the and(0.1 Pht-HrTH (n=12), before acutely inhibition prepulse 1+MK-801 (n=12) ( (n=15), Pht-HrTH vehicle administering Figure 7 group).MK-801 alone ***p<0.001 (**p<0.01, withthecompared control group; compared with the ###p<0.001 (PPI) inWistar rats. The data are indicated themeansas ± thestandard error of meanthe test before andLia the prepulse acutely inhibition 801 mg/kg) (n=12), 1+MK-801 (0.1 (n=12) Lia-AKH ( (n=15), vehicle administering Figure 6 mean (*p<0.05, ***p<0.001 **p<0.01, withcompared the controlgroup). test (PPI)inhibition The rats. in data are indicated Wistar as ±the means standard errorof the prepulse the before (n=12) acutely and Ani1+MK-801 (n=12), mg/kg) (0.1 MK-801(n=12), vehicleadministering (n=15), Ani-AKH ( Figure 5 compared with the group). MK-801-alone mean

(*p<0.05, **p<0.01 compared with the control group; #p<0.05, ##p<0.01, ###p<0.001 ##p<0.01, (*p<0.05,**p<0.01with the control group; compared #p<0.05, the illustrates data on prepulse inhibitionPPI) (i.p.) by (% intraperitoneally obtained illustrates the illustrates data on prepulse inhibitionPPI) (i.p.) by (% intraperitoneally obtained the illustrates data on prepulse inhibitionPPI) (i.p.) by (% intraperitoneally obtained Libellula Auripennis Libellula Phormia-Terrae Anax ImperatorMauricianus HrTH) MK-801 (1 (n=12), mg/kg) AKH) MK- (n=12), (1 mg/kg) AKH) (1 mg/kg) AKH) (1mg/kg) Accepted Article This by is protectedarticle reserved. Allrights copyright. Fig 1a Figures

Total distance moved (m) 100 20 40 60 80 0

C o ntr o l

Ani-AKH AAPA test AAPA Drugs (mg/kg) Drugs

2 M K -80 1

A 0 *** n .1 i-A 5 KH 2+ M K-801 #

Accepted Article This by is protectedarticle reserved. Allrights copyright. Fig 1b Fig 1c Total distance moved (m) Total distance moved (m) 100 100 20 40 60 80 20 40 60 80 0 0

Control C ontrol

Pht- Lia-AK H AAPA test AAPA AAPA test AAPA Drugs (mg/kg) Drugs rTH 2 (mg/kg) Drugs H 2

M M K K-80 -801 0.15 1 Lia-AKH 0 P .1 ** h * t-H 5 rTH+MK 2+MK-801

-801 **

Accepted Article This by is protectedarticle reserved. Allrights copyright. Fig 2a Fig 2b Number of entries Number of entries 15 20 25 10 15 20 25 10 0 5 0 5

C C ontr o nt o ro l l

Li A n a i-A -AK Drugs (mg/kg) Drugs (mg/kg) Drugs AAPA test AAPA K test AAPA H H 2 2 MK M K -80 -801 0. 1 Li 0 Ani-AKH 2+MK-80 ** a .1 ** 1 -A 5 5 K H 2+ M K

- ### 80 ## 1 1

Accepted Article This by is protectedarticle reserved. Allrights copyright. Fig 2c Fig 3a Number of shocks Number of entries 15 20 25 20 40 60 80 10 0 5 0

C C o ontr nt ro ol l

A Pht n i-A -H Drugs (mg/kg) Drugs (mg/kg) Drugs KH test AAPA rT test AAPA H 2 2 M MK K -80 -80 1 1 Ani 0 P 0

ht ** .15 ** .15 - -H AKH rTH 2+MK 2+MK

-80 - ###

8 ## 01 1

Accepted Article This by is protectedarticle reserved. Allrights copyright. Fig 3b Fig 3c Number of shocks Number of shocks 60 80 20 40 60 80 20 40 0 0

Control C ontr o l

Pht-HrTH 2 Li a -AK Drugs (mg/kg) Drugs (mg/kg) Drugs AAPA test AAPA test AAPA H 2 MK-80 MK -80 1 1 0.1 Li 0 P ** .1 a * ht-HrTH+MK-8015 -A 5 K H 2+ M K -80 ## ## 1

Accepted Article Fig 4b This by is protectedarticle reserved. Allrights copyright. Fig 4a Maximum time of avoidance (s) Maximum time of avoidance (s) 1000 1000 1500 600 800 500 200 400 0 0

Cont Contr

rol o l

Li A a n -A i-A AAPA test AAPA test AAPA

Drugs (mg/kg) Drugs K (mg/kg) Drugs KH H 2 2 MK-801 MK- 801 Lia-AKH 0 Ani-AKH 2+MK-801 0. . **

1 * 1 5 5

2 +MK

-801 ### ##

Accepted Article Fig 5 This by is protectedarticle reserved. Allrights copyright. Fig 4c

%PPI Maximum time of avoidance (s) 10 20 30 40 50 0 1000 200 400 600 800 0

C o n trol

Drugs (mg/kg) Drugs Pht-H PPI test * AAPA test AAPA Drugs (mg/kg) Drugs rT H 2 MK *** -80 1 0 Pht- .1 * 5 H rTH+MK ***

-801 #

MK-801+Ani-AKH 1 MK-801 AKHAni 1 Vehiculum

Accepted Article This by is protectedarticle reserved. Allrights copyright. Fig 7 Fig 6

%PPI %PPI -20 30 40 50 10 20 20 40 60 0 0 Drugs (mg/kg) Drugs (mg/kg)Drugs PPI test PPI test ** ** *** ** ### MK-801+Pht-HrTH 1 MK-801 Pht-HrTH 1 Vehiculum MK-801+Lia-AKH 1 MK-801 1 Lia-AKH Vehiculum