THE CANNABINOID AGONIST HU 210 MODIFIES RAT BEHAVIOURAL RESPONSES TO NOVELTY AND STRESS

Pharmacological Research, Vol. 41, No. 1, 2000 Article No. phrs.1999.0560, available online at http:rrwww.idealibrary.com on

THE CANNABINOID AGONIST HU 210 MODIFIES RAT BEHAVIOURAL RESPONSES TO NOVELTY AND STRESS D. GIULIANI, F. FERRARIU and A. OTTANI Department of Biomedical Sciences, Di¨ ision of Pharmacology, Uni¨ ersity of Modena, ¨ ia G. Campi 287, I-41100, Modena, Italy Accepted 7 June 1999

Experiments were performed on groups of rats after acute and sub-chronic treatment Žonce daily for 9 days. with the cannabinoid agonist HU 210 Ž25᎐100 ␮ g kgy1 , i.p.. as well as 24 h and 7 days after the last drug injection. The animals underwent three behavioural tests in novel environments. In the observation cages ŽTest 1., rat locomotor activity was found to be dose-dependently reduced after acute and sub-chronic treatment at all doses and virtually unchanged during abstinence; grooming was potently inhibited by acute treatment but potentiated by the sub-chronic one at doses of 50 and 100 ␮ g kgy1 , the effect of the higher dose persisting after 24 h and 7 days abstinence. Vocalization in animals in response to a tactile stimulus was highest after HU 210 at 100 ␮ g kgy1 in all experimental modes except after 7 days abstinence. In the X-maze ŽTest 2., sub-chronic HU 210 dosedependently enhanced rat natural aversion for open arms, and this behaviour persisted during abstinence after the highest dose. Grooming in the X-maze was completely absent in rats acutely injected with HU 210 but potentiated in those sub-chronically treated or abstinent. In the swimming test ŽTest 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 the whole, our results are indicative of an anxiogenic-like effect of sub-chronic HU 210 at high doses and reflect the persistence of enhanced emotional response to novel environments 䊚 2000 Academic Press when the treatment is discontinued. KEY

WORDS:

cannabinoids, HU 210, anxiety-like state, rat.

INTRODUCTION It is well established that the psychotropic effects of ⌬9-tetrahydrocannabinol Ž ⌬9-THC., the major active component of marijuana w1x, are linked to changes in the activity of central neurotransmitter systems w2᎐4x. The discovery of specific central cannabinoid binding sites ŽCB1. w5᎐8x which seem to be primarily involved in many of the behavioural and physiological alterations produced in humans and animals by ⌬9-THC w9᎐13x, has led to the synthesis of a number of new cannabinoids w10, 12x. Of those, HU 210 has demonstrated a high degree of correlation between its ability to bind to CB1 receptors and its efficacy in producing in ¨ i¨ o effects w13᎐16x. Several clinical and experimental findings have promoted the present study on HU 210. It has been reported that anxiogenic reactions are elicited by marijuana or U

Corresponding author.

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⌬9-THC in humans, particularly after high doses and long-term exposure w17᎐19x; similarly, an anxiety-like state has been found in cannabinoid-treated rodents subjected to different behavioural models w16, 20, 21x. Again, it has been demonstrated that ⌬9-THC whether acutely or sub-chronically administered, potently stimulates adrenocorticotropin hormone ŽACTH. secretion w22x, and that central corticotropin-releasing factor ŽCRF. systems play a key role in the mediation of cannabinoid-induced anxiogenic effects w16x. ACTH and CRF vigorously enhance self-grooming in the rat w23, 24x and this seems to represent a response to a state akin to psychological human stress, since it manifests itself as stereotyped behaviour in different stressful situations. There appear to be few reports on the influence of cannabinoids on grooming. Some researchers have observed no significant modification w25x, while others have demonstrated inhibition induced by acute HU 210 w15x or ⌬9-THC w26x. 䊚 2000 Academic Press

48

The aim of our work was, therefore, to investigate in more detail the influence exerted by HU 210 on rat grooming and on general animals’ behaviour in novel anxiogenic environments w27᎐29x. Rats’ behavioural patterns were recorded after acute and sub-chronic treatment with the drug at different doses, as well as during abstinence. In addition, other parameters that fulfil the criteria for evaluating cannabimimetic activity, namely, locomotor activity w10x and vocalization w30x, were evaluated in some experimental conditions.

MATERIALS AND METHODS

Animals and general beha¨ ioural procedure The subjects were male SPF-Wistar rats ŽHarlan Nossan, Udine, Italy. weighing 200᎐230 g at the outset. They were housed in groups of four with food 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 the start of the experiments. The experiments were performed between 09:00 and 14:00 h in a soundproof, air-conditioned room, with normal lighting conditions, where the animals were monitored by trained observers unaware of the treatment schedule. The controls were handled in the same way as the treated animals and received vehicle injections. Experimentally-naive rats were randomly assigned to various groups that were submitted to three tests in three experimental conditions, namely, after acute treatment with HU 210 Ž25, 50 or 100 ␮ g kgy1 , i.p.., sub-chronic treatment Žonce daily for 9 days at the same doses. and 24 h after the last injection Žabstinence.. Each rat performed one test only and was used only in one experimental condition, thus avoiding the risk that memory of the environment might influence its behaviour. Having observed that, after 24 h abstinence, the rats injected with 100 ␮ g kgy1 behaved in a significantly different manner from the controls in all tests, other naive animals were subchronically treated at this dose and, 7 days after the last injection, underwent Tests 1 and 2 Žsee below.. The in-force regulations on the care of animals for scientific purposes ŽCEE Concil 86r603; Italian D.L. 27-01-92 N⬚ 116. were strictly complied with. Test 1: locomotor acti¨ ity, grooming and ¨ ocalization in the obser¨ ation cages Behavioural evaluations were carried out on groups of animals Žhomogeneous as regards treatment . which were transferred to glass observation cages Ž40 = 30 = 34 cm. 50 min after the i.p. injection of HU 210 or vehicle Žacute and sub-chronic ., as well as during abstinence. The test Ž30 min. started immediately after the animals had been placed in the observation cages and the parameters considered were locomotor activity, grooming and vocalization. Locomotor acti¨ ity was scored as described elsewhere w31x, each

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rat being observed for 30 s at 5-min intervals and rated on a scale 0᎐2 where: 0 s absent, 1 s discontinuous locomotor activity Žexploratory behaviour, rearing and lateral movements., 2 s uninterrupted locomotor activity for at least 25 s. This evaluation allowed the contemporaneous assessment of grooming. Grooming was evaluated according to Gispen et al. w23x. In brief, an observer recorded at 15-s intervals whether or not each rat displayed the phenomenon, defined as face and body washing, scratching, licking paws or tail. If one of these signs was observed a positive score was given. Locomotor activity and grooming values for each rat are the sum of all the scores attributed to the animal for each parameter during the test period. Immediately after their removal from the cages, the same animals were tested for the presence or absence of ¨ ocalization. For this purpose, each rat was gently pressed two to four times bilaterally behind its forelimb, on the ventral aspect of the frontal costal region, the experimenter using thumb and forefinger w30x. Test 2: X-maze A test was performed on animals which were placed singly into the X-maze, 60 min after the i.p. injection of HU 210 or vehicle Žacute and sub-chronic ., as well as during abstinence. The apparatus was as previously described w32x. In brief, it consists of a black Plexiglas floor in the form of a regular cross, the arms of which are flanked by transparent Plexiglas walls and meet at the intersection to form a central arena; one opposite pair of arms is covered, while the other is left open. Each rat was gently put into the central arena of the apparatus, facing an open arm, and its subsequent exploratory behaviour was timed over a 5-min period. A rat was taken to have entered an arm when all four paws were on it. The number of open- and closed-arm entries, the time spent in each arm and the number of total entries were recorded. Total grooming displayed during the test was counted with a stopwatch. At the end of each test, the animal was removed and the maze floor was thoroughly cleaned. Test 3: swimming-test This test was performed on animals which were placed singly into a swimmingpool, 60 min after the i.p. injection of HU 210 or vehicle Žacute and sub-chronic ., as well as during abstinence. Swimming strategy was observed for 2 min in a circular tank Ž130 cm in diameter, 60 cm in depth., constructed of white Plexiglas that, prior to testing, had been filled to a depth of 45 cm with water maintained at 30 " 2⬚C. The experimenters recorded the episodes of wall-hugging, defined as a complete turn of the pool in contact with the wall w29x.

Drugs and treatments HU 210 ŽTocris-Cookson, Bristol, UK. was freshly prepared as a suspension containing a drop of Tween

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80 Ž0.1%. and distilled water at concentrations that allowed the injection of 1 ml kgy1 intraperitoneally Ži.p... The doses of HU 210 and the pretreatment time were chosen on the basis of preliminary experiments w29, 33x.

Statistical e¨ aluation Data for locomotor activity and grooming, presented as means " SEM of the cumulative values of the two parameters obtained for each rat in the test period, were analysed using, as appropriate: Kruskal᎐Wallis test followed by Mann᎐Whitney Utest. As for vocalization, only its presence or absence was noted and the percentage of animals vocalizing was analysed using ANOVA followed by Student᎐Newman᎐Keuls test. Data for X-maze test are presented as means " SEM; the number of entries into the open arms is shown as a percentage of the total number of arm entries, and the time spent in the open arm is similarly given as a percentage of the total time spent in the arms. Grooming values are the means " SEM of the cumulative seconds spent in the Xmaze. All parameters, as well as the number of total entries, were analysed by the ANOVA followed by Student᎐Newman᎐Keuls test and Student’ t-test. Data for the swimming test are presented as number of animals displaying wall-hugging per group; chi-squared test was used. The level of significance was set at P- 0.05. At least six animals were used in each treatment group, the exact number of rats is reported in the figures and tables.

RESULTS

Test 1: locomotor acti¨ ity, grooming and ¨ ocalization in the obser¨ ation cages Results obtained in Test 1 are reported in Fig. 1. Acute and sub-chronic cannabinoid Ž25᎐100 ␮ g kgy1 , i.p.. dose-dependently decreased locomotor activity wFig. 1Ža.x, but the inhibitory effect obtained with the highest dose was significantly lower after sub-chronic than after acute treatment. At no dose was any significant modification of this parameter observed after 24 h abstinence with respect to the controls. While acute HU 210 potently inhibited rat grooming, sub-chronic treatment enhanced it, the effect persisting after 24 h abstinence for the doses of 50 and 100 ␮ g kgy1 wFig. 1Žb.x. The percentage of rats vocalizing increased dose-dependently in all three experimental modes wFig. 1Žc.x. Table I shows locomotor activity, grooming and vocalization displayed by rats sub-chronically treated with HU 210 at 100 ␮ g kgy1 , 7 days after the last injection. While locomotor activity and vocalization were similar to those of vehicle-treated animals, grooming was found to be higher.

Fig. 1. Effect of HU 210 on locomotor activity, grooming and vocalization in rats. HU 210 ŽHU 25᎐100 ␮ g kgy1 . or vehicle were i.p. injected 50 min before the observation period Ž30 min.. Each histogram is the mean " SEM of the cumulative scores Ža, locomotor activity; b, grooming. for each rat or the percentage of animals vocalizing Žc, vocalization.. Number of rats used: acute treatments, eight animals for all groups; sub-chronic treatments, vehicle s 6, HU 25 s 8, HU 50 s 8, HU 100 s 6; abstinence, vehicle s 8, HU 25 s 8, HU 50 s 11, HU 100 s 7. ŽU . Significantly different from the respective vehicle-treated rats; Ž'. significantly different from acutely treated rats at the same dose; ŽKruskal᎐Wallis followed by Mann᎐Whitney U-test.. ŽB. Significantly different from the respective vehicle treated rats ŽANOVA followed by Student᎐ Newman᎐Keuls test..

Test 2: X-maze Figure 2Ža᎐c. shows the results obtained in rats undergoing the X-maze test. Vehicle-injected rats always exhibited an adversion for open arms wFig.

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Table I Effect of HU 210 (100 ␮g kg I1 ) on locomotor activity, grooming and vocalization in test 1, 7 days after last injection

Vehicle HU 210

Motor acti¨ ity (score)

Grooming (score)

Vocalization (%)

7.7" 0.1 7.5" 0.2

2.5" 0.4 8.0" 1.5

12 25

HU 210 Ž100 ␮ g kgy1 . or vehicle were i.p. injected. Each value for locomotor activity or grooming is the mean " SEM of the cumulative scores for each rat during the observation period Ž30 min.; the percentage of vocalizing animals is shown. Eight animals were used for each group. U P- 0.05 ¨ s vehicle ŽMann᎐Whitney U-test..

2Ža,b.x. However, it was observed that the controls tested after sub-chronic treatment and during abstinence spent more time in the open arms than those treated acutely ŽF2,15 s 12.9, Ps 0.000. wFig. 2Žb.x. Acute HU 210 seemed to reduce the permanence into open arms, with respect to controls, only at the dose of 25 ␮ g kgy1 wFig. 2Ža,b.x, although significance was not reached; no result was obtained with the other two doses wFig. 2Ža,b.x, probably owing to a state of marked hypoactivity wFig. 2Žc.x that randomly froze the animals in the arms. After subchronic treatment there was a significant reduction in the number of open arm entries after doses of 50 and 100 ␮ g kgy1 ŽF3,20 s 4.5, Ps 0.01. wFig. 2Ža.x, as there was in the time spent there at all three dosage levels ŽF3,20 s 8.4, Ps 0.000. wFig. Ž2b.x. After 24 h abstinence, there was a reduction in the number of open arm entries ŽF3,20 s 5.1, Ps 0.009. wFig. 2Ža.x and in the time spent there ŽF3,20 s 4, Ps 0.02. wFig. 2Žb.x in the animals treated with the cannabinoid at the dose of 100 ␮ g kgy1 . The number of total entries wFig. 2Žc.x decreased dose-dependently after acute HU 210 treatment ŽF3,20 s 16.8, Ps 0.000. and, to a lesser extent, after sub-chronic treatment ŽF3,20 s 5.8, Ps 0.005. but remained similar to that of vehicle-treated rats during abstinence. Comparison of the influence exerted on rats by the same dose of HU 210 in each of the three experimental modes Žacute, sub-chronic or abstinence., showed remarkable differences among the groups. At the dose of 25 ␮ g kgy1 , the time spent in the open arms after sub-chronic treatment and during abstinence was higher than after acute treatment ŽF2,15 s 8.9, Ps 0.003. wFig. 2Žb.x. A similar difference occurred for total arm entries ŽF2,15 s 7, Ps 0.007. wFig. 2Žc.x: at the dose of 50 ␮ g kgy1 , total arm entries during abstinence were significantly highest than after acute and sub-chronic treatment ŽF2,15 s 16.2, Ps 0.000., as they were at 100 ␮ g kgy1 ŽF2,15 s 63.8, Ps 0.000.. Grooming ŽFig. 3., virtually non-existent in all but a few of the acutely-treated controls, was completely

Fig. 2. Effect of HU 210 on X-maze behaviour in rats. HU 210 ŽHU 25᎐100 ␮ g kgy1 or vehicle were i.p. injected 60 min before the test Ž5 min.. Each histogram is the mean " SEM of the percentage of open arm entries Ža., time spent there Žb. or number of total entries Žc.. Number of rats used: six animals for all groups; ŽU . Significantly different from the respective vehicle treated rats; Ž'. significantly different from acutely-treated rats at the same dose; Ž䢇. significantly different from abstinent rats at the same dose ŽANOVA followed by Student᎐ Newman᎐Keuls test..

abolished by acute HU 210 at all doses ŽF3,20 s 8.2, Ps 0.000. but increased after sub-chronic cannabinoid ŽF3,20 s 7.6, Ps 0.001. and during abstinence ŽF3,20 s 6.1, Ps 0.004.. Here, too, the same dose of HU 210 affected the rats differently, depending on the experimental mode: after sub-chronic treatment at all three doses grooming was higher than in the other two groups ŽF2,15 s 6.1, Ps 0.01., ŽF2,15 s 13, Ps 0.000. and ŽF2,15 s 8.2, Ps 0.004., respectively. Table II shows the X-maze performance displayed by rats sub-chronically treated with HU 210 at the dose of 100 ␮ g kgy1 , 7 days after the last injection. While total entries were similar to those of vehicle-

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Fig. 3. Effect of HU 210 on grooming in the X-maze. Animals and treatments as in Fig. 2. Each histogram is the mean " SEM of the cumulative time displayed by each rat during the test period Ž5 min.. Ž). Significantly different from the respective vehicle-treated rats; Ž'. significantly different from acutely-treated rats at the same dose; Ž䢇. significantly different from abstinent rats at the same dose ŽANOVA followed by Student᎐Newman᎐Keuls test..

treated rats, the number of open arm entries and the time spent there were significantly reduced Ž t s 4.9, Ps 0.000; t s 4.9, Ps 0.000, respectively. and grooming was potentiated Ž t s 4.8, Ps 0.000..

Test 3: swimming test Table III shows that, after acute treatments, all groups behaved similarly in their swimming strategy; in the sub-chronic mode, rat treated with 50 and 100 ␮ g kgy1 followed an enhanced thigmotactic pattern Ž ␹ 2 s 12.6; Ps 0.007.. The phenomenon was confirmed during 24 h abstinence Ž ␹ 2 s 10.7; Ps 0.01..

DISCUSSION Our results shed further light on the behavioural effects of HU 210 w16, 13x, in particular on the anxiogenic-like properties of this potent synthetic cannabinoid w21x. As expected, when acutely administered to rats, the drug dose-dependently produced sedation and reduced locomotion, as also occurs after ⌬9-THC and other cannabinoids w20x. In general, tolerance, more or less complete, has been

demonstrated for most of the pharmacological effects of cannabinoids w15, 20x, and in the present study partial tolerance for depression of locomotor activity was confirmed. In addition, comparison of the inhibitory effects on this parameter in Test 1 Žlocomotor activity. and Test 2 Žnumber of total entries in the X-maze. showed that the phenomenon of tolerance strictly depends on the dose and the experimental model used. In fact, as already pointed out w34x, it is practically impossible to obtain the same results in behavioural models that elicit different emotional states in the animal. Although the animals acutely injected with 50 and 100 ␮ g kgy1 of HU 210 exhibited marked sedation, they were hypersensitive to tactile stimuli and vocalized strongly when touched. Vocalization is considered a pointer of cannabimimetic activity w30x, and it is elicited by ⌬9-THC at doses much higher than those of HU 210. Since, at the doses of 25 and 50 ␮ g kgy1 HU 210 has proved to be analgesic in rats Ždata not yet published. the possibility that vocalization might be due to pain can be excluded, rather, it seems to reflect enhanced irritability in response to tactile stimulus. In accordance with other authors w20x, we

Table II Effect of HU 210 (100 ␮g kg I1 ) on X-maze test, 7 days after last injection Treatments

Open arms Entries (%)

Vehicle HU 210

Time spent Total entries Grooming (%) (no.) (s)

37.5" 2.7 23.5" 2.6 11.5" 1.0 0 13.8" 3.5U 7.7" 1.9U 9.5" 0.7 21.8" 4.5U

HU 210 or vehicle were i.p. injected. Each value is the mean " SEM of eight animals, during the observation period Ž5 min.. U P- 0.05 ¨ s vehicle ŽStudent t-test..

Table III Effect of HU 210 on swimming test Treatment (␮ g kg y 1) Vehicle HU 210, 25 RU 210, 50 HU 210, 100

Wall-hugging Acute

Sub-chronic

Abstinence

2r8 1r8 4r8 4r8

1r8 0r8 5r8 6r8U

1r8 1r8 5r8 6r8U

HU 210 or vehicle were i.p. injected. Data are presented as number of animals displaying wall-hugging during the test period Ž2 min.. U P- 0.05 ¨ s vehicle Žchi-squared test..

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therefore hypothesize that vocalization, like aggressiveness, which is frequently reported in cannabinoid-treated rodents subjected to stress-inducing procedures w16, 20x, is a behavioural expression of heightened emotionality associated with a state of fear. The correlation between cannabinoids and stress has long been proposed w35, 36x and supported by experimental findings on animals, namely, that cannabinoids induce a potent secretion of ACTH w22x and CRF w16x, which, as is known, play a key role in stress w37, 23x. Moreover, the attenuation exerted by the CRF antagonist D-phenyl CRF12᎐41 on rat anxiogenic responses to HU 210 w16x strongly suggests the mediation of endogenous CRF systems in these effects. Increased rat grooming, which has been recognized as occurring under certain mildly-stressful events, has been found to be markedly potentiated by central administration of CRF w24x. CRF releases ACTH from the pituitary and ACTH also stimulates grooming w23x. Our findings that grooming is enhanced after sub-chronic HU 210 at 50 and 100 ␮ g kgy1 is therefore not surprising. In addition, we observed a grooming syndrome during abstinence after the highest dose of the compound. In contrast with the present data, which suggest a behavioural sensitization, grooming has been found to be depressed by acute HU 210 while remaining unaffected in sub-chronically-treated rats, in which the drug merely developed a behavioural tolerance to this decrease w15x. The hypothesis that the grooming enhancement observed after sub-chronic treatment simply depends on a phenomenon of tolerance to the sedative effects of the drug is not tenable. In fact, sub-chronic treatment at 25 and 50 ␮ g kgy1 did not produce an effect significantly different from that after acute treatment on locomotor activity, which remained significantly different from that of vehicle-treated rats; however, in these two groups enhancement of grooming was evident. At the dose of 25 ␮ g kgy1 , grooming was higher than that after acute treatment; at that of 50 ␮ g kgy1 it was also higher than that observed in vehicle-treated rats. The dose of 100 ␮ g kgy1 produced a partial tolerance to the inhibition of locomotor activity, which remained lower than that of vehicle-treated rats. In this case too, grooming was much higher than that of vehicle-treated rats and not simply the same, as would occur if the phenomenon were strictly dependent on tolerance to sedative effects. While caution must be exercised in attributing emotional responses to specific animal behavioural patterns, our results in the X-maze test also confirm that, after sub-chronic treatment with certain doses of HU 210 and during abstinence, the rats displayed an anxiety-like state. The X-maze test has already

been used to highlight anxiogenic-like effects of several cannabinoids, and acute HU 210, in particular, has been found to intensify animals’ aversion to the open arms w21x. No significant change in X-maze behaviour has been seen by the authors during withdrawal, for any of the compounds tested. In the present study sub-chronically treated and abstinent rats were consistently different in their behaviour from vehicle-injected animals, and the parameters that were found to be modified Žgrooming and exploration of novel environments. anthropomorphically reflect an anxiogenic response. Aversion for open spaces and wall-hugging, as persistence in trying to escape along the walls of the pool, have been attributed to an anxiety-like state w27᎐29x. Our data on vocalization, in line with those reported for ⌬9 THC w25x, apparently contradict a supposed enhancement of fear response in tolerant and abstinent animals, for vocalization was slightly higher after acute HU 210 injection than in the other two experimental modes. At present we are not in a position to say whether tolerance is responsible for this phenomenon. It might be simply ascribable to the fact that our acutely-injected animals were unused to any handling, unlike those in the other two groups, for adaptation to handling seems to be the main explanation for the less anxious behaviour of subchronically-treated control rats in the X-maze test. In conclusion, in view of the number of similarities between animal and human behaviour after cannabinoids, our study suggests an anxiogenic activity of the potent CB1 agonist HU 210, after subchronic treatment at high doses, and the persistence of enhanced emotional response to novel environments when the drug is discontinued.

ACKNOWLEDGEMENTS This work was supported by grants from Ministero della Pubblica Istruzione Ž60%. and by CNR.

REFERENCES 1. Gaoni Y, Mechoulam R. Isolation, structure and partial synthesis of an active constituent of hashish. J Am Chem Soc 1964; 86: 1646᎐7. 2. Bloom AS. Effect of ⌬9-tetrahydrocannabinol on the synthesis of dopamine and norepinephrine in mouse brain synaptosomes. J Pharmacol Exp Ther 1982; 221: 97᎐103. 3. Poddar MK, Biswas B, Ghosh JJ. ⌬9-THC and brain biogenic amines. In: Drugs and central synaptic transmission. Bradley PB, Dhawan BN eds. London: MacMillan, 1976: 193᎐9. 4. Tripathi HL, Vocci FJ, Dewey WL. Effect of cannabinoids on cholinergic systems in various regions of the mouse brain. Fed Proc 1979; 38: 590.

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

5. Devane WA, Dysarz FAIII, Johnson MR, Melvin LS, Howlett AC. Determination and characterization of a cannabinoid receptor in rat brain. Mol Pharmacol 1988; 34: 605᎐13. 6. Harris LS, Carchman RA, Martin BR. Evidence for the existence of specific cannabinoid binding sites. Life Sci 1978; 22: 1131᎐8. 7. Herkenham M, Lynn AB, Johnson MR, Melvin LS, De Costa BR, Rice KC. Characterization and localisation of cannabinoid receptors in rat brain: a quantitative in ¨ itro autoradiographic study. J Neurosci 1991; 11: 563᎐83. 8. Nye JS, Seltzman HH, Pitt CG, Snyder SH. Highaffinity cannabinoid binding sites in brain membranes labelled with w 3 Hx-5⬘-trimethylammonium Ž ⌬8-THC.. J Pharmacol Exp Ther 1985; 234: 784᎐91. 9. Collins DR, Pertwee RG, Davies SN. The action of synthetic cannabinoids on the induction of long-term potentiation in the rat hippocampal slice. Eur J Pharmacol 1994; 259: R7᎐8. 10. Compton DR, Johnson MR, Melvin LS, Martin B. Pharmacological profile of a series of bicyclic cannabinoid analogs: classification as cannabimimetic agents. J Pharmacol Exp Ther 1992; 260: 201᎐9. 11. Compton DR, Rice KC, De Costa BR, Razdan RK, Melvin LS, Johnson MR, Martin BR. Cannabinoid structure ᎐activity relationship: correlation of receptor binding and in ¨ i¨ o activities. J Pharmacol Exp Ther 1993; 265: 218᎐26. 12. Little PJ, Compton DR, Johnson MR, Melvin SL, Martin BR. Pharmacology and stereoselectivity of structurally novel cannabinoids in mice. J Pharmacol Exp Ther 1988; 247: 1046᎐51. 13. Little PJ, Compton DR, Mechoulam R, Martin BR. Stereochemical effects of 11-OH-⌬8-THCdimethyleptyl in mice and dogs. Pharmacol Biochem Beha¨ 1989; 32: 661᎐6. 14. Howlett AC, Champion TM, Wilken GH, Mechoulam R. Stereochemical effects of 11-OH-delta 8tetrahydrocannabinol-dimethylheptyl to inhibit adenylate cyclase and bind to the cannabinoid receptor. Neuropharmacology 1990; 29: 161᎐5. 15. De Fonseca FR, Calderon JL, Mechoulam R, Navarro M. Repeated stimulation of D1 dopamine receptors enhances Žy.-11-hydroxy-D8-tetrahydrocannabinoldimethylheptil-induced catalepsy in male rats. Neuroreport 1994; 5: 761᎐5. 16. De Fonseca FR, Rubio P, Menzaghi F, Merlo-Pich E, Rivier J, Koob GF, Navarro M. Corticotropinreleasing factor ŽCRF. antagonist wD-PHE12, Nle21,38, CaMeLeu37xCRF attenuates the acute actions of the highly potent cannabinoid receptor agonist HU 210 on defensive-withdrawal behaviour in rats. J Pharmacol Exp Ther 1996; 276: 56᎐64. 17. Fishman MW, Rosenbaum JF, Yabusaki DI, Carr DB. Marijuana related anxiety: a questionnaire-based pilot study of normal and psychiatric populations. Res Commun Subst Abuse 1988; 9: 219᎐26. 18. Weil AT, Zinberg NE, Nelsen JM. Clinical psychological effects of marijuana in man. Science (Wash. DC) 1968; 162: 1234᎐42. 19. Zuardi AW, Shirakawa I, Finkelford E, Karniol IG. Action of cannabinol on the anxiety and other effects produced by ⌬9-THC in normal subjects. Psychopharmacolology 1982; 76: 245᎐50. 20. Dewey WL. Cannabinoid pharmacology. Pharmacol

53

Re¨ 1986; 38: 151᎐78. 21. Onaivi ES, Green MR, Martin BR. Pharmacological characterization of cannabinoids in the elevated plus maze. J Pharmac Exp Ther 1990; 253: 1002᎐9. 22. Dewey WL, Peng TC, Harris LS. The effect of 1-trans⌬9 THC on the hypothalamohypophyseal-adrenal axis of rats. Eur J Pharmacol 1970; 12: 382᎐4. 23. Gispen WH, Wiegant HM, Greven HM, De Wied D. The induction of excessive grooming in the rat by intraventricular application of peptides derived from ACTH-structure activity studies. Life Sci 1975; 17: 645᎐50. 24. Morley JE, Levine AS. Corticotrophin releasing factor, grooming and ingestive behaviour. Life Sci 1982; 31: 1459᎐64. 25. Jarbe TUC, Hiltunen AJ. Cannabimimetic activity of cannabinol in rats and pigeons. Neuropharmacology 1987; 26: 219᎐28. 26. Romero J, Garcia L, Cebeira M, Zadrozny D, Fernandez-Ruiz JJ, Ramos JA. The endogenous cannabinoid receptor ligand, anandamide, inhibits the motor behaviour: role of nigrostriatal dopaminergic neurons. Life Sci 1995; 56: 2033᎐40. 27. Pellow S, Chopin P, File SE, Briley M. Valuation of open-closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J Neurosci Meth 1985; 14: 149᎐67. 28. Sutherland RJ, Rudy JW. Place learning in the Morris place navigation task is impaired by damage to the hippocampal formation even if the temporal demands are reduced. Psychobiology 1988; 16: 157᎐63. 29. Ferrari F, Ottani A, Giuliani D. Learning impairment produced in rat by the cannabinoid agonist HU 210 in a water maze task. Pharmacol Bioch Beha¨ 1999: in press. 30. Henriksson BG, Jarbe TUC. Cannabis-induced vocalization in the rat. J Pharm Pharmacol 1971; 23: 457᎐8. 31. Ferrari F, Giuliani D. Influence of eticlopride on cocaine- and DA D 2 agonist-induced behavioural effects in rats. Pharmacol Biochem Beha¨ 1996; 53: 523᎐30. 32. Ferrari F, Tartoni PL, Mangiafico V. B-HT 920 antagonizes rat neophobia in the X-maze test: a comparative study with other drugs active on adrenergic and dopaminergic receptors. Arch Int Pharmacodyn Ther 1989; 298: 7᎐14. 33. Ferrari F, Ottani A, Giuliani D. Cannabinoid activity in rats and pigeons of HU 210, a potent anti-emetic drug. Pharmacol Biochem Beha¨ 1999; 62: 75᎐80. 34. Ferrari F, Giuliani D. Effect on rat feeding behaviour of two selective D 2 dopamine agonists. Physiol Beha¨ 1994; 56: 921᎐6. 35. Kumar MS, Chen CL. Effect of an acute dose of ⌬9-tetrahydrocannabinol on hypothalamic luteinizing hormone releasing hormone and met-enkephalin content and serum levels of testosterone and corticosterone in rats. Subst Alcohol Actionsr Misuse 1983; 4: 37᎐43. 36. Maclean KI, Littleton JM. Environmental stress as a factor in the response of rat brain catecholamine metabolism to ⌬9 THC. Eur J Pharmacol 1977; 41: 171᎐82. 37. Dunn AJ, File SE. Corticotropin-releasing factor has an anxiogenic action on the social interaction test. Horm Beha¨ 1987; 21: 193᎐202.