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Pharmacological Research, Vol. 41, No. 1, 2000 Article No. phrs.1999.0560, available online at http: rr www.idealibrary.com onTHE CANNABINOID AGONIST HU 210 MODIFIES RAT BEHAVIOURALRESPONSES TO NOVELTY AND STRESSD. GIULIANI, F. FERRARI U and A. OTTANIDepartment of Biomedical Sciences, Di ision of Pharmacology, Uni ersity of Modena,ia G. Campi 287, I-41100, Modena, Italy Accepted 7 June 1999Experiments were performed on groups of rats after acute and sub-chronic treatment oncedaily for 9 days . with the cannabinoid agonist HU 210 25 100 g kg y1 , i.p. . as well as 24 hand 7 days after the last drug injection. The animals underwent three behavioural tests innovel environments. In the observation cages Test 1 . , rat locomotor activity was found tobe dose-dependently reduced after acute and sub-chronic treatment at all doses and

virtually unchanged during abstinence; grooming was potently inhibited by acute treatmentbut potentiated by the sub-chronic one at doses of 50 and 100 g kg y1, the effect of thehigher dose persisting after 24 h and 7 days abstinence. Vocalization in animals in responseto a tactile stimulus was highest after HU 210 at 100 g kg y1 in all experimental modesexcept after 7 days abstinence. In the X-maze Test 2 . , sub-chronic HU 210 dose-dependently enhanced rat natural aversion for open arms, and this behaviour persistedduring abstinence after the highest dose. Grooming in the X-maze was completely absent inrats acutely injected with HU 210 but potentiated in those sub-chronically treated or abstinent. In the swimming test Test 3 . rats sub-chronically treated at 50 and 100 pg kgy1 displayed relevant wall-hugging and the same occurred 24 h after last injection. On thewhole, our results are indicative of an anxiogenic-like effect of sub-chronic HU 210 at highdoses and reect the persistence of enhanced emotional response to novel environmentswhen the treatment is discontinued. 2000 Academic PressK EY WORDS: cannabinoids, HU 210, anxiety-like state, rat.INTRODUCTIONIt is well established that the psychotropic effects of 9 -tetrahydrocannabinol 9 -THC . , the major activecomponent of marijuana w 1 x , are linked to changes inthe activity of central neurotransmitter systems w 2 4 x .The discovery of specic central cannabinoid bind-ing sites CB1 .w 5 8 x which seem to be primarily

involved in many of the behavioural and physiologi-cal alterations produced in humans and animals by9 -THC w 9 13 x , has led to the synthesis of a number of new cannabinoids w 10, 12 x . Of those, HU 210 hasdemonstrated a high degree of correlation betweenits producing ability to in bind i o to effects CB 1receptors w 13 and its efcacy in16 x . Several clinical

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and experimental ndings have promoted the pre-sent study on HU 210. It has been reported thatanxiogenic reactions are elicited by marijuana or UCorresponding author.9-THC in humans, particularly after high doses and

long-term exposure w 17 19 x ; similarly, an anxiety-likestate has been found in cannabinoid-treated rodentssubjected to different behavioural models w 16, 20,21 x . Again, it has been demonstrated that 9 -THCwhether acutely or sub-chronically administered, po-tently stimulates adrenocorticotropin hormone ACTH . secretion w 22 x , and that central corti-cotropin-releasing factor CRF . systems play a keyrole in the mediation of cannabinoid-induced anxio-genic effects w 16 x . ACTH and CRF vigorously en-hance self-grooming in the rat w 23, 24 x and this

seems to represent a response to a state akin topsychological human stress, since it manifests itself as stereotyped behaviour in different stressful situa-tions.There appear to be few reports on the inuenceof cannabinoids on grooming. Some researchers haveobserved no signicant modication w 25 x , while oth-ers have demonstrated inhibition induced by acuteHU 210 w 15 x or 9 -THC w 26 x .1043 6618 r 00r 010047 07 r $35.00 r 0 2000 Academic Press

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Pharmacological 48Research, Vol. 41, No. 1, 2000 The aim of our work was, therefore, to investigatein more detail the inuence exerted by HU 210 onrat grooming and on general animals behaviour innovel anxiogenic environments w 27 29 x . Rats be-havioural patterns were recorded after acute andsub-chronic treatment with the drug at differentdoses, as well as during abstinence. In addition,other parameters that full the criteria for evaluat-ing cannabimimetic activity, namely, locomotor ac-tivity w 10 x and vocalization w 30 x , were evaluated insome experimental conditions.MATERIALS AND METHODS

Animals and general beha ioural procedureThe subjects were male SPF-Wistar rats HarlanNossan, Udine, Italy . weighing 200 230 g at theoutset. They were housed in groups of four withfood and water ad libitum and on a 12-h light cycle,from 07:00 to 19:00 h, for at least 1 week prior to thestart of the experiments. The experiments were per-formed between 09:00 and 14:00 h in a soundproof,air-conditioned room, with normal lighting condi-tions, where the animals were monitored by trainedobservers unaware of the treatment schedule. Thecontrols were handled in the same way as the treatedanimals and received vehicle injections. Experimen-tally-naive rats were randomly assigned to variousgroups that were submitted to three tests in threeexperimental conditions, namely, after acute treat-ment with HU 210 25, 50 or 100 g kg y1 , i.p. . ,sub-chronic treatment once daily for 9 days at thesame doses . and 24 h after the last injection absti-nence . . Each rat performed one test only and wasused only in one experimental condition, thus avoid-ing the risk that memory of the environment mightinuence its behaviour. Having observed that, after

24 h abstinence, the rats injected with 100 g kg y1behaved in a signicantly different manner from thecontrols in all tests, other naive animals were sub-chronically treated at this dose and, 7 days after thelast injection, underwent Tests 1 and 2 see below . .The in-force regulations on the care of animals for scientic purposes CEE Concil 86 r 603; Italian D.L.27-01-92 N 116 . were strictly complied with.

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Test 1: locomotor acti ity, grooming and ocaliza -tion in the obser ation cages Behavioural evalua-tions were carried out on groups of animals homo-geneous as regards treatment . which were trans-ferred to glass observation cages 40=30=34 cm. 50 min after the i.p. injection of HU 210 or vehicle acute and sub-chronic . , as well as during absti-nence. The test 30 min . started immediately after the animals had been placed in the observationcages and the parameters considered were locomo-tor activity, grooming and vocalization. Locomotor acti ity was scored as described elsewhere w 31 x , eachrat being observed for 30 s at 5-min intervals andrated on a scale 0 2 where: 0 sabsent, 1 sdiscontinuous locomotor activity exploratory be-haviour, rearing and lateral movements . , 2 suninterrupted locomotor activity for at least 25 s.

This evaluation allowed the contemporaneous as-sessment of grooming. Grooming was evaluated ac-cording to Gispen et al. w 23 x . In brief, an observer recorded at 15-s intervals whether or not each ratdisplayed the phenomenon, dened as face and bodywashing, scratching, licking paws or tail. If one of these signs was observed a positive score was given.Locomotor activity and grooming values for eachrat are the sum of all the scores attributed to theanimal for each parameter during the test period.Immediately after their removal from the cages, thesame animals were tested for the presence or absence of ocalization. For this purpose, each ratwas gently pressed two to four times bilaterallybehind its forelimb, on the ventral aspect of thefrontal costal region, the experimenter using thumband forenger w 30 x .Test 2: X-maze A test was performed on animalswhich were placed singly into the X-maze, 60 minafter the i.p. injection of HU 210 or vehicle acuteand sub-chronic . , as well as during abstinence. Theapparatus was as previously described w 32 x . In brief,it consists of a black Plexiglas oor in the form of a

regular cross, the arms of which are anked bytransparent Plexiglas walls and meet at the intersec-tion to form a central arena; one opposite pair of arms is covered, while the other is left open. Eachrat was gently put into the central arena of theapparatus, facing an open arm, and its subsequentexploratory behaviour was timed over a 5-min pe-riod. A rat was taken to have entered an arm when

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all four paws were on it. The number of open- andclosed-arm entries, the time spent in each arm andthe number of total entries were recorded. Totalgrooming displayed during the test was counted witha stopwatch. At the end of each test, the animal wasremoved and the maze oor was thoroughly cleaned.Test 3: swimming-test This test was performed onanimals which were placed singly into a swimming-pool, 60 min after the i.p. injection of HU 210 or vehicle acute and sub-chronic . , as well as duringabstinence. Swimming strategy was observed for 2min in a circular tank 130 cm in diameter, 60 cm indepth . , constructed of white Plexiglas that, prior totesting, had been lled to a depth of 45 cm withwater maintained at 30 "2 C. The experimentersrecorded the episodes of wall-hugging, dened as acomplete turn of the pool in contact with the wall

w 29 x .Drugs and treatmentsHU 210 Tocris-Cookson, Bristol, UK . was freshlyprepared as a suspension containing a drop of Tween

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Pharmacological Research, Vol. 41, No. 1, 2000 4980 0.1% . and distilled water at concentrations thatallowed the injection of 1 ml kg y1 intraperitoneally i.p. . . The doses of HU 210 and the pretreatmenttime were chosen on the basis of preliminary experi-ments w 29, 33 x .Statistical e aluationData for locomotor activity and grooming, pre-sented as means "SEMof the cumulative values of the two parameters obtained for each rat in the testperiod, were analysed using, as appropriate:

Kruskal Wallis test followed by Mann Whitney U -test. As for vocalization, only its presence or absencewas noted and the percentage of animals vocalizingwas analysed using ANOVA followed byStudent Newman Keuls test.Data for X-maze test are presented as means "SEM; the number of entries into the open arms isshown as a percentage of the total number of armentries, and the time spent in the open arm issimilarly given as a percentage of the total timespent in the arms. Grooming values are the means

"SEMof the cumulative seconds spent in the X-maze. All parameters, as well as the number of totalentries, were analysed by the ANOVA followed byStudent Newman Keuls test and Student t -test.Data for the swimming test are presented as num-ber of animals displaying wall-hugging per group;chi-squared test was used.The level of signicance was set at P -0.05. Atleast six animals were used in each treatment group,the exact number of rats is reported in the guresand tables.RESULTSTest 1: locomotor acti ity, grooming and o-calization in the obser ation cagesResults obtained in Test 1 are reported in Fig. 1.Acute and sub-chronic cannabinoid 25 100 gkg y1 , i.p. . dose-dependently decreased locomotor

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activity w Fig. 1 a .x , but the inhibitory effect obtainedwith the highest dose was signicantly lower after sub-chronic than after acute treatment. At no dosewas any signicant modication of this parameter observed after 24 h abstinence with respect to thecontrols. While acute HU 210 potently inhibited ratgrooming, sub-chronic treatment enhanced it, theeffect persisting after 24 h abstinence for the dosesof 50 and 100 g kg y1 w Fig. 1 b .x . The percentage of rats vocalizing increased dose-dependently in allthree experimental modes w Fig. 1 c .x .Table I shows locomotor activity, grooming andvocalization displayed by rats sub-chronically treatedwith HU 210 at 100 g kg y1, 7 days after the lastinjection. While locomotor activity and vocalizationwere similar to those of vehicle-treated animals,grooming was found to be higher.

Fig. 1. Effect of HU 210 on locomotor activity, groomingand vocalization in rats. HU 210 HU 25 100 g kg y1. or vehicle period 30 were min i.p. . . injected 50 Each histogram min before the is the mean "observationSEMof thecumulative each rat scores a, or the percentage locomotor activity; b, of animals vocalizing grooming c, . vocal-for ization . . Number of rats used: acute treatments, eightanimals for all groups; sub-chronic treatments, vehicle s6,HU 8, HU 25 s8, 25 s8, HU HU 50 s8, 50 s11, HU 100 s6; HU 100 s7. abstinence, U.Signicantlyvehicle sdifferent from the respective vehicle-treated rats; '. signicantly different from acutely treated rats at thesame dose; Kruskal Wallis followed by Mann WhitneyU -test . . B .Signicantly different from the respectivevehicle treated rats ANOVA followed by StudentNewman Keuls test . .Test 2: X-mazeFigure 2 a c . shows the results obtained in ratsundergoing the X-maze test. Vehicle-injected ratsalways exhibited an adversion for open arms w Fig.

LOCOMOTOR ACTIVITYScore 8 M

Vehicle

A

GROOMING

A

A

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Vehicle HU 25 HU 50

(2)

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Pharmacological 50Research, Vol. 41, No. 1, 2000 Table IEffect of HU 210 ( 100 g kg I1 ) on locomotor activity,grooming and vocalization in test 1, 7 days after lastinjectionMotor acti ity Grooming Vocalization( score ) ( score ) ( %) Vehicle 7.7 "0.1 2.5 "0.4 12HU 210 7.5 "0.2 8.0 "1.5 25HU 210 100 g kg y1. or vehicle were i.p. injected.Each value for locomotor activity or grooming is themean "SEMof the cumulative scores for each rat duringthe observation period 30 min . ; the percentage of vocaliz-ing animals is shown. Eight animals were used for eachgroup.U P -0.05 s vehicle Mann Whitney U -test . .2 a,b .x . However, it was observed that the controlstested after sub-chronic treatment and during absti-nence spent more time in the open arms than thosetreated Acute acutely HU 210 seemed F 2,15s12.9, to P s0.000 .w Fig. reduce the permanence2 b .x .into open arms, with respect to controls, only at thedose of 25 g kg y1 w Fig. 2 a,b .x , although signi-

cance was not reached; no result was obtained withthe other two doses w Fig. 2 a,b .x , probably owing to astate of marked hypoactivity w Fig. 2 c .x that ran-domly froze the animals in the arms. After sub-chronic treatment there was a signicant reductionin the number of open arm entries after doses of 50and 100 g there was in kg the y1 time F 3,20spent s4.5, there P s0.01 at .w Fig. 2 a .x , asall three dosagelevels abstinence, F 3,20s8.4, there P s0.000 .w Fig. 2b .x . was a reduction in the After 24 hnumber of

open and 2 b .x in in arm the the time entries animals spent F treated 3,20there s5.1, with F 3,20P s0.009 s4, the .w Fig. 2 a.x P s0.02 .w Fig.cannabinoid atthe dose of 100 g kg y1. The number of totalentries w Fig. 2 c .x decreased dose-dependently after acute HU 210 and, to a lesser extent, treatment after F sub-chronic 3,20

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other two P s0.000 . Table II and groups shows F 2,15the F s8.2, 2,15 X-maze s6.1, P s0.004 performance P s0.01 . , . , respectively.F 2,15s13,displayedby rats sub-chronically treated with HU 210 at thedose of 100 g kg y1, 7 days after the last injection.While total entries were similar to those of vehicle-

ENTRIES 1N OPEN ARMS (a)

Vehicle HU 25

TIME SPENT IN OPEN ARMS

Vehicle HU 25

TOTAL ENTRIES

A A

Acute H Sub-chronic E Abstinence

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Fig. 3. mean "Effect of SEM

of the HU 210 on grooming in cumulative time displayed the X-maze. by each rat Animals during the test and period treatments 5 min as . . ) in . Fig. Signicantly 2. Each histogram is different from thetherespective vehicle-treated different from abstinent rats; rats at the ' . same signicantly dose ANOVA different followedfrom acutely-treated by Student rats Newman at the Keuls same test . .dose; . signicantlytreated rats, the number of open arm entries andthe time spent there were signicantly reduced t s4.9, P s0.000; t s4.9, P s0.000, respectively . andgrooming was potentiated t s4.8, P s0.000 . .Test 3: swimming test Table III shows that, after acute treatments, allgroups behaved similarly in their swimming strategy;in the sub-chronic mode, rat treated with 50 and 100g kgy1 followed an enhanced thigmotactic pattern 2 s12.6; P s0.007 . . The phenomenon was con-rmed during 24 h abstinence 2 s10.7; P s0.01 . .DISCUSSIONOur results shed further light on the behaviouraleffects of HU 210 w 16, 13 x , in particular on theanxiogenic-like properties of this potent syntheticcannabinoid w 21 x . As expected, when acutely admin-istered to rats, the drug dose-dependently produced

sedation and reduced locomotion, as also occursafter 9 -THC and other cannabinoids w 20 x . In gen-eral, tolerance, more or less complete, has beenTable IIEffect of HU 210 ( 100 g kg I1 ) on X-maze test, 7 daysafter last injectionTreatments Open armsEntries Time spent Total entries Grooming ( % ) ( % ) ( no. ) () sVehicle HU 210 37.5 "2.7 13.8 "3.5 U 23.5"2.6 7.7 "1.9 U 11.5 "1.0 09.5"0.7 21.8 "4.5U HU 210 or vehicle were i.p. injected. Each value is themean "SEMdemonstrated for most of the pharmacological ef-fects of cannabinoids w 15, 20 x , and in the presentstudy partial tolerance for depression of locomotor activity was conrmed. In addition, comparison of the inhibitory effects on this parameter in Test 1 locomotor activity . and Test 2 number of totalentries in the X-maze . showed that the phenomenon

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of tolerance strictly depends on the dose and theexperimental model used. In fact, as already pointedout w 34 x , it is practically impossible to obtain thesame results in behavioural models that elicit dif-ferent emotional states in the animal. Although theanimals acutely injected with 50 and 100 g kg y1 of HU 210 exhibited marked sedation, they were hy-persensitive to tactile stimuli and vocalized stronglywhen touched. Vocalization is considered a pointer of cannabimimetic activity w 30 x , and it is elicited by9-THC at doses much higher than those of HU 210.Since, at the doses of 25 and 50 g kg y1 HU 210has proved to be analgesic in rats data not yetpublished . the possibility that vocalization might bedue to pain can be excluded, rather, it seems toreect enhanced irritability in response to tactilestimulus. In accordance with other authors w 20 x , we

Table IIIEffect of HU 210 on swimming testTreatment ( g kg y1 ) Wall-hugging Acute Sub-chronic AbstinenceVehicle 2 r 8 1r 8 1r 8HU 210, 25 1 r 8 0r 8 1r 8RU 210, 50 4 r 8 HU 210, 100 4 r 8 5r 8 6r 8 U 5r 86r 8U HU 210 or vehicle were i.p. injected. Data are pre-sented as number of animals displaying wall-hugging dur-ing U the P -0.05 test period s of eight animals, during the observation pe-riod U P -0.05 5 min . .

s vehicle Student t -test . . 2 min . .vehicle chi-squared test . .

Pharmacological Research, Vol. 41, No. 1, 2000 51

Seconds

GROOMING

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Pharmacological 52Research, Vol. 41, No. 1, 2000 therefore hypothesize that vocalization, like aggres-siveness, which is frequently reported in cannabi-noid-treated rodents subjected to stress-inducingprocedures w 16, 20 x , is a behavioural expression of heightened emotionality associated with a state of fear.The correlation between cannabinoids and stresshas long been proposed w 35, 36 x and supported byexperimental ndings on animals, namely, thatcannabinoids induce a potent secretion of ACTHw 22 x and CRF w 16 x , which, as is known, play a keyrole in stress w 37, 23 x . Moreover, the attenuation

exerted by the CRF antagonistDbeen used to highlight anxiogenic-like effects of several cannabinoids, and acute HU 210, in particu-lar, has been found to intensify animals aversion tothe open arms w 21 x . No signicant change in X-mazebehaviour has been seen by the authors during with-drawal, for any of the compounds tested.In the present study sub-chronically treated andabstinent rats were consistently different in their behaviour from vehicle-injected animals, and theparameters that were found to be modied groom-ing and exploration of novel environments . an-thropomorphically reect an anxiogenic response.-phenyl CRF12 41on rat anxiogenic responses to HU 210 w 16 x stronglysuggests the mediation of endogenous CRF systemsin these effects.Increased rat grooming, which has been recog-nized as occurring under certain mildly-stressfulevents, has been found to be markedly potentiatedby central administration of CRF w 24 x . CRF releasesACTH from the pituitary and ACTH also stimulates

grooming w 23 x . Our ndings that grooming is en-hanced after sub-chronic HU 210 at 50 and 100 gkgy1 is therefore not surprising. In addition, weobserved a grooming syndrome during abstinenceafter the highest dose of the compound. In contrastwith the present data, which suggest a behaviouralsensitization, grooming has been found to be de-pressed by acute HU 210 while remaining unaf-fected in sub-chronically-treated rats, in which the

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drug merely developed a behavioural tolerance tothis decrease w 15 x .The hypothesis that the grooming enhancementobserved after sub-chronic treatment simply de-pends on a phenomenon of tolerance to the sedativeeffects of the drug is not tenable. In fact, sub-chronictreatment at 25 and 50 g kg y1 did not produce anAversion for open spaces and wall-hugging, as per-sistence in trying to escape along the walls of thepool, have been attributed to an anxiety-like statew 27 29 x .Our data on vocalization, in line with those re-ported for 9 THC w 25 x , apparently contradict a sup-posed enhancement of fear response in tolerant andabstinent animals, for vocalization was slightly higher after acute HU 210 injection than in the other twoexperimental modes. At present we are not in a

position to say whether tolerance is responsible for this phenomenon. It might be simply ascribable tothe fact that our acutely-injected animals were un-used to any handling, unlike those in the other twogroups, for adaptation to handling seems to be themain explanation for the less anxious behaviour of subchronically-treated control rats in the X-mazetest.In conclusion, in view of the number of similari-ties between animal and human behaviour after cannabinoids, our study suggests an anxiogenic activ-ity of chronic the potent treatment at CB high 1agonist HU 210, after sub-doses, and the persistenceof enhanced emotional response to novel environ-ments when the drug is discontinued.effect signicantly different from that after acutetreatment on locomotor activity, which remainedsignicantly different from that of vehicle-treatedACKNOWLEDGEMENTSrats; however, in these two groups enhancement of grooming was evident. At the dose of 25 g kg y1,grooming was higher than that after acute treat-

This work was supported by grants from Ministerodella Pubblica Istruzione 60% . and by CNR.ment; at that of 50 g kg y1 it was also higher thanthat observed in vehicle-treated rats. The dose of 100 g kg y1 produced a partial tolerance to theREFERENCESinhibition of locomotor activity, which remainedlower than that of vehicle-treated rats. In this case

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