Cannabinoids Modulate Hippocampal Memory and Plasticity

8/11/2019 Cannabinoids Modulate Hippocampal Memory and Plasticity

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Cannabinoids Modulate Hippocampal Memory and Plasticity

Hila Abush and Irit Akirav*

ABSTRACT: Considerable evidence demonstrates that cannabinoidagonists impair whereas cannabinoid antagonists improve memory andplasticity. However, recent studies suggest that the effects of cannabi-noids on learning do not necessarily follow these simple patterns, partic-ularly when emotional memory processes are involved. We investigatedthe involvement of the cannabinoid system in hippocampal learning andplasticity using the fear-related inhibitory avoidance (IA) and the non-fear-related spatial learning paradigms, and cellular models of learningand memory, i.e., long-term potentiation (LTP) and long-term depression(LTD). We found that microinjection into the CA1 of the CB1/CB2 re-ceptor agonist WIN55,212-2 (5 lg/side) and an inhibitor of endocanna-binoid reuptake and breakdown AM404 (200 ng/side) facilitated theextinction of IA, while the CB1 receptor antagonist AM251 (6 ng/side)impaired it. WIN55,212-2 and AM251 did not affect IA conditioning,while AM404 enhanced it, probably due to a drug-induced increase inpain sensitivity. However, in the water maze, systemic or local CA1injections of AM251, WIN55,212-2, and AM404 all impaired spatiallearning. We also found that i.p. administration of WIN55,212-2(0.5 mg/kg), AM404 (10 mg/kg), and AM251 (2 mg/kg) impaired LTP inthe Schaffer collateral-CA1 projection, whereas AM404 facilitated LTD.Our findings suggest diverse effects of the cannabinoid system on CA1memory and plasticity that cannot be categorized simply into an impair-ing or an enhancing effect of cannabinoid activation and deactivation,respectively. Moreover, they provide preclinical support for the sugges-tion that targeting the endocannabinoid system may aid in the treatmentof disorders associated with impaired extinction-like processes, such aspost-traumatic stress disorder. VVC 2009 Wiley-Liss, Inc.

KEY WORDS: CB1 receptors; extinction; LTP; LTD; avoidance; CA1

INTRODUCTION

Learning and memory impairments are among the most commonlyreported behavioral effects of cannabinoids (Lichtman et al., 1995; Pam-plona and Takahashi, 2006). These effects are thought to be associatedwith the hippocampus, an area highly expressed with cannabinoid recep-tors (Berrendero et al., 1999). Systemic administration of cannabinoidagonists induces deficits in several hippocampal dependent tasks, such asthe radial and water maze (Iwasaki et al., 1992; Lichtman et al., 1995;Ferrari et al., 1999; Varvel et al., 2001; Da Silva and Takahashi, 2002).Furthermore, direct administration of cannabinoid receptor agonists into

the hippocampus affects performance in the radial maze and delayedalternation in the T-maze (Lichtman et al., 1995; Egashira et al., 2002;Suenaga and Ichitani, 2004), suggesting that hippocampal CB1 receptors

are involved in learning and memory processes. Onthe other hand, studies using CB1 receptor antago-nists or CB1-deficient mice usually demonstrateincreased memory performance in hippocampal-dependent memory tasks like the radial maze(Lichtman, 2000) and social recognition (Terranovaet al., 1996).

Similarly, previous studies indicate that cannabinoidreceptor activation in hippocampal slices impairs long-term potentiation (LTP; Nowicky et al., 1987; Collinset al., 1994; Terranova et al., 1995) and long-termdepression (LTD; Misner and Sullivan, 1999), whereasCB1 blockade enhances LTP in hippocampal slices,

and a similar enhancement of LTP is observed inmice lacking cannabinoid CB1 receptors (Bohmeet al., 2000; Slanina et al., 2005).

Taken together, considerable evidence demonstratesthat cannabinoid agonists impair whereas cannabinoidantagonists improve memory and plasticity (forreview, Riedel and Davies, 2005). However, recentstudies suggest that the effects of cannabinoids onlearning do not necessarily follow these previouslydescribed simple patterns, particularly when emotionalmemory processes are involved (Chhatwal and Ressler,2007; Lutz, 2007).

An increasing number of studies demonstrate thatthe cannabinoid system is critical for the encoding ofemotional memory and for the extinction of fear-related memories (Marsicano et al., 2002; Milad andQuirk, 2002; Chhatwal et al., 2005; Laviolette andGrace, 2006a,b; Viveros et al., 2007). CB1-deficientmice show impaired auditory fear extinction, withunaffected memory acquisition and consolidation(Marsicano et al., 2002). Additionally, there arereports of facilitated fear extinction following systemiccannabinoid activation (Chhatwal et al., 2005; Pam-plona et al., 2006, 2008) although several reportsfailed to show accelerated extinction following the

administration of cannabinoid agonists WIN55,212-2and tetrahydrocannabinol (THC; Chhatwal et al.,2005; Kobilo et al., 2007; Varvel et al., 2007).

Thus, in the present set of experiments, we aimedto study whether emotional and nonemotional mem-ory formation and two models of synaptic plasticityare differentially modulated by the cannabinoid sys-tem. Hence, we examined the effects of exogenouslyadministered cannabinoid receptor agonist and antag-onist on a hippocampal-dependent fear-related mem-ory paradigm [i.e., inhibitory avoidance (IA)] and anon-fear-based paradigm (i.e., spatial learning). In

Department of Psychology, University of Haifa, Haifa, IsraelGrant sponsor: The National Institute for Psychobiology, Israel; Grantnumber: 203-07-08 (to I.A.).*Correspondence to: Irit Akirav, PhD, Department of Psychology, Univer-sity of Haifa, Haifa 31905, Israel. E-mail: [email protected] for publication 20 August 2009DOI 10.1002/hipo.20711Published online 14 October 2009 in Wiley Online Library(wileyonlinelibrary.com).

HIPPOCAMPUS 20:11261138 (2010)

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addition, we examined the effects of these drugs on LTP andits functional inverse LTD in the Schaffer collateral-CA1 path-way using the intact rat, rather than acute hippocampal slices,as the experimental model.

MATERIALS AND METHODS

Subjects

Male SpragueDawley rats (60 days old, 250300 g) werecaged individually at 22 6 28C under 12-h light/dark cycles(lights turned on at 07:00 and turned off at 19:00). Rats hadaccess to water and laboratory rodent chow ad libitum. Theexperiments were approved by the University of Haifa Ethicsand Animal Care Committee, and adequate measures weretaken to minimize pain or discomfort in accordance with theguidelines laid down by the National Institutes of Health in

the US regarding the care and use of animals for experimentalprocedures.Except for the sensory-motor sequence tests, every rat under-

went one behavioral test or electrophysiological test, with thedifferent drugs (or drug doses) to prevent carryover effects dueto multiple behavioral tests.

Drug Treatment

The synthetic full CB1/2-receptor agonist WIN55,212-2(WIN), an inhibitor of endocannabinoid reuptake and break-down AM404, and the CB1 receptor antagonist AM251(Tocris, USA) were initially dissolved in dimethylsulfoxide

(DMSO) and further diluted with saline (0.9% NaCl). FinalDMSO concentration was


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Another set of rats implanted bilaterally with cannula intothe CA1 were anesthetized and perfused transcardially with200 ml of saline, followed by 200 ml of 4% paraformaldehyde(PFA), pH 7.4. The brains were removed and then immersedin 30% sucrose in PBS until they sank. Coronal sections(60 lm) were cut using a freezing cryostat, mounted on gela-tin-coated glass slides, and stained with methylene blue. Slideswere examined under a light microscope. Figure 1b shows pho-tomicrograph illustrating a typical cannula track in CA1 ofrepresentative brain section. As we used a small volume of

infusion (0.5 ll), the possibility of injection spread to otherhippocampal subfields is little.

Sensory-Motor Tests

Three tests to investigate sensory-motor parameters were pre-sented to the rats in sequence: an open field, an elevated plus-maze, and a pain sensitivity tests (see below). All tests wereconducted in dimly lit rooms. Drugs were microinjected intothe CA1 20 min before the rats began the three-test sequence.The order of the tests was counterbalanced between the groups:half of the rats first experienced the open field test, while theother half began with the elevated plus-maze. The pain sensitiv-ity test was always third in the sequence, because it involved afootshock that could otherwise have affected performance inthe open field and maze tasks. The open field and elevated plusmaze tests were conducted for 5 min each, with 5-min acclima-tion to the test chamber prior to testing.

Open fieldThe open field consisted of a closed wooden box. The walls

were painted black and the floor was white and divided by 1-cm-wide black lines into 25 squares measuring 10 3 10 cm2

each. A video image of the entire open field was displayed on aTV monitor, and the movements of the rat, which was initiallyplaced in a corner of the field, were manually recorded and an-alyzed to measure motor activity over a period of 5 min.

Recordings were made of the time the rat spent in the centraland the peripheral squares, the number of instances of rearing,and the total distance covered. The open field arena was thor-oughly cleaned between each trial with odorous clean wipes.

Elevated plus-maze

The elevated plus-maze consisted of four arms (65 cm each)on a stand 50 cm high. The floor of the maze was white. Twoof the arms had no railing (open arms), and the other twoarms were protected by opaque railings (painted black) to aheight of 35 cm (closed arms).

The rats were individually placed at the center of the maze,facing an open arm. Subsequently, the time that the rat spentin each of the different arms, and the number of entries intothe closed and open arms were manually recorded for a periodof 5 min, and the percent of each parameter was calculated.

Pain sensitivity

Pain sensitivity was assessed by determining the footshock in-tensity (mA) that elicited a discomfort response (i.e., flinch orvocalization) (Kim et al., 1991). Rats were individually placedin a Plexiglas box (25 3 25 3 34 cm3) with a floor consistingof 13 stainless steel rods of diameter 5 mm, spaced every 1 cm.Each rat received a continuously ascending mild electric foot-shock (beginning at 0.0 mA and ending as soon as the animalshowed discomfort) via the metal grid floor to determine cur-rent thresholds at which each animal would exhibit a flinch ora vocalization response. Two observers independently scored

FIGURE 1. Schematic drawing of cannulae tip positions in theCA1 area of the hippocampus. (a) Schematic drawings of CA1 can-nulae placements. Shown is a coronal view at position 4.16 and4.3 mm posterior to bregma. Solid black circles indicate the loca-tion of cannulae. (b) Photomicrograph illustrating a typical can-nula track in CA1 of a representative brain section (about 4.16mm posterior to bregma). The full arrow represents the position ofthe tip of the cannula and the dashed arrow illustrates where theposition of the injection cannula would be (extending 1 mmbeyond the tip of the cannula into the CA1).

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flinch and vocalization thresholds. We found that all the ratsshowed a flinch response that was sometimes accompanied by avocalization response. Thus, the flinch response determined thethreshold.

Electrophysiology

Surgical procedure

Rats were anesthetized (with 40% urethane, 5% chloralhydrate in saline, injection volume of 4 ml/1 kg, i.p.) andplaced in a stereotaxic frame. Small burr holes were drilled inthe skull to allow electrodes to be inserted into the brain. A re-cording microelectrode (glass, tip diameter of 25 lm, filledwith 2 M NaCl, resistance of 14 M) was slowly lowered intothe CA1 area (anteroposterior, 24.2 mm; lateral, 62.5 mm;ventral, 22 to 3 mm). A bipolar 125-lm stimulating electrodewas positioned to activate the Schaffer collateral-CA1 projec-tion (anteroposterior, 23.1 mm; lateral, 60.4 mm; ventral,2

3.5 mm). After positioning the electrodes, the rat was left for60 min before commencing the experiment.

LTP induction

LTP was induced by u-like high-frequency stimulation(HFS) to the Schaffer collateral (three sets of 10 trains; eachtrain consisting of 10 pulses at 200 Hz; intertrain interval,200 ls; interset interval, 1 min). Field potentials were recordedfrom the CA1 every 5 min for 90 min after HFS to theSchaffer collateral. LTP was measured as an increase in the am-plitude of the excitatory postsynaptic potentials (EPSPs). Poten-tiation was measured as a percentage change from the average

of the 30 min baseline before HFS.

LTD induction

LTD was elicited by low-frequency stimulation (LFS) to theSchaffer collateral (4 Hz). Field potentials were recorded fromthe CA1 every 5 min for 90 min after LFS to the Schaffer col-lateral. LTD was measured as a decrease in EPSP amplitude.Depression was measured as a percentage change from the aver-age of the 30 min baseline before LFS.

In both experiments, evoked responses were digitized(10 kHz) and analyzed using the Cambridge Electronic Design(Cambridge, UK) 14011 and its Spike 2 software. Offline

measurements were made of EPSP amplitude using averages offive successive responses to a given stimulation intensity appliedat 0.1 Hz (Akirav and Richter-Levin, 2002).

Drugs were i.p. injected 20 min (vehicle, WIN55,212-2, andAM404) or 30 min (AM251, see Sink et al., 2008) beforeapplying HFS or LFS to the Schaffer collateral.

The Morris water maze task

The maze was placed in a dimly lit room. Animals wereindividually placed at several start locations around the mazeand were required to find the location of a hidden platform

within 60 s. If a rat did not reach the platform within 60 s, anexperimenter would guide it there (Roozendaal et al., 2004).The rat was then allowed to remain on the platform for 25 sbefore removal back to the home cage, to enable the rat tolearn its location from the spatial cues around the maze.

The experiment consisted of an acquisition day, on which

the animals went through a massed protocol of 14 trials(1 min each, with a 3-min rest between trials) (Akirav et al.,2001), and a test day (24 h later), on which the rats weretested for their ability to locate the escape platform (1-mintrial). Drugs were either injected i.p. or microinjected into theCA1, 30 min before the first acquisition trial.

Statistical Analysis

The results are expressed as means 6 SEM. For statisticalanalysis, repeated-measures ANOVA for treatment 3days (IA),treatment 3 blocks (spatial learning), or treatment 3 time(electrophysiology) was used. One-way ANOVA was used tocompare between the different drug treatments. t-test was usedin the water maze task. All post-hoc comparisons were madeusing the least significant difference multiple-comparison test(LSD).

Experimental Design

Inhibitory avoidance

Eighty-five rats were implanted with cannulae into the CA1and left 1 week to recuperate. Drugs or vehicle were microin-

jected into the CA1 20 min before IA conditioning (Pre-Cond)or 20 min before the first extinction trial (pre-Ext1). Each rat

was microinjected once.

Electrophysiology

A second group of rats (n 5 81) was anesthetized and takenfor electrophysiological recording in the CA1. Drugs or vehiclewere i.p. injected 2030 min prior to HFS or LFS. In anotherset of animals (n 5 20), drugs or vehicle were i.p. injectedwith no HFS or LFS (Fig. 4b).

Spatial learning

Thirty-two rats were implanted with cannulae into the CA1and left 1 week to recuperate. Drugs or vehicle were microin-jected into the CA1 30 min before spatial training in the watermaze. In another set of rats (n 5 34), drugs or vehicle werei.p. injected 30 min before spatial training in the water maze.

Sensory-motor parameters

Twenty-seven rats were implanted with cannulae into theCA1 and left 1 week to recuperate. Drugs or vehicle weremicroinjected into the CA1 20 min before the first sensory-motor test (the open field or the elevated plus-maze).

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RESULTS

The Effects of Cannabinoid Activation andDeactivation in the CA1 on InhibitoryAvoidance Conditioning and Extinction

To examine whether the CB1 receptor in the CA1 is essen-tial in IA conditioning and extinction, rats were microinjectedwith the CB1 receptor antagonist AM251 (6 ng/0.5 ll) eitherbefore conditioning (Cond) or before the first extinction trial(Ext1). Repeated measures ANOVA [treatment 3 days (4 35)] revealed a significant difference between the groups in termsof their latencies to enter the dark side of the box (F(3,29) 5

4.59, P 5 0.009; Fig. 2a). The within-subject latencies did notdiffer significantly between the days (F(1,29) 5 2.57, NS), butthere was a significant interaction effect (F(3,29) 5 5.26, P 50.005).

Post-hoc comparison unveiled a significant differencebetween the group microinjected with AM251 prior to Ext1

(Preext1-AM251, n 5 8) and all the other groups, i.e., thevehicle groups (Preext1-Vehicle, n 5 10: P 5 0.004; Precond-Vehicle, n 5 8: P 5 0.005) and the group microinjected with

AM251 prior to conditioning (Precond-AM251, n 5 7: P 50.006). One-way ANOVA on the different days of the experi-ment revealed that the significant main effect stemmed from adifference between the latencies of the groups on Ext3 (F(3,29)5 4.05, P 5 0.016) and Ext4 (F(3,29) 5 7.97, P 5 0.01).Post-hoc comparison revealed that the latency of the Preext1-

AM251 group was significantly greater than that of the othergroups (Ext3: Precond-Vehicle: P 5 0.005; Preext1-Vehicle: P5 0.006; Precond-AM251: P 5 0.023) (Ext4: Precond-Vehi-cle: P < 0.001; Preext1-Vehicle: P < 0.001; Precond-AM251:

P 5 0.001). Thus, AM251 had no effect on IA acquisitionwhen microinjected before conditioning, but impaired extinc-tion when microinjected before the first extinction trial.

Following the clear result we obtained from using the CB1receptor antagonist to block IA extinction, we next askedwhether stimulation of cannabinoid receptor signaling mightaccelerate the extinction rate. A previous report (Chhatwalet al., 2005) showed WIN55,212-2 and AM404 to have differ-ent effects on extinction; hence, both agonists were tested here.

WIN 55,212-2 (5 lg/0.5 ll) or AM404 (200 ng/0.5 ll)were microinjected into the CA1 20 min before the first extinc-tion trial (Ext1). Repeated measures ANOVA [treatment 3days (4 3 5)] revealed a significant difference between thegroups in terms of their latencies to enter the dark side of the

FIGURE 2. The effects of cannabinoid receptor antagonist andagonists into the CA1 on inhibitory avoidance conditioning andextinction. (a) AM251 (6 ng/0.5 ll) microinjected into the CA1before the first extinction trial (Preext1-AM251, n 5 8) signifi-cantly increased the latency of the rats to enter the dark side ofthe box compared with the vehicle groups (Preext1-Vehicle, n 510; Precond-Vehicle, n 5 8) and with the group that received amicroinjection of AM251 prior to conditioning (Precond-AM251,n 5 7), indicating the blockade of extinction (Ext3: $P < 0.05,Preext1-AM251 differs from Precond-AM251; #P < 0.01, Preext1-

AM251 differs from the vehicle groups) (Ext4: P < 0.001, Preext1-

AM251 differs from all the groups). (b) WIN55,212-2 (5 lg/0.5 ll) or AM404 (200 ng/0.5 ll) microinjected into the CA1before the first extinction trial (Preext1-WIN, n 5 9; Preext1-

AM404, n 5 8) significantly decreased the latency of the rats toenter the dark side of the box compared with the vehicle group(Preext1-Vehicle,n 512) on Ext1 and Ext2, indicating facilitationof extinction (P < 0.01, vehicle differs from all the groups). (c)

AM404 (200 ng/0.5 ll) microinjected into the CA1 before condi-tioning (Precond-AM404, n 5 7) significantly increased the la-tency of the rats to enter the dark side of the box compared withthe vehicle and WIN groups (Precond-Vehicle, n 5 8, Precond-

WIN, n 5 8) on Ext2-Ext4 (Ext2 and Ext4: P < 0.05, Precond-AM404 differs from all the groups, Ext3: P < 0.01, Precond-AM404 differs from all the groups).

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box (F(2,26) 5 5.92, P 5 0.008; Fig. 2b). The within-subjectlatencies also differed significantly between the days (F(1,26) 53.92, P 5 0.051), but there was no interaction effect (F(2,26)5 1.44, NS). Post-hoc comparison unveiled a significant differ-ence between the vehicle group (Preext1-Vehicle, n 5 12) andthe groups microinjected with WIN (Preext1-WIN, n 5 9:

P 50.007) or AM404 (Preext1-AM404, n 58: P 50.009).One-way ANOVA on the different experimental days revealed

that the significant main effect stemmed from a difference in la-tency between the groups on Ext1 (F(2,26) 5 6.32, P 5 0.006)and Ext2 (F(2,26) 5 7.29, P 5 0.003). Post-hoc comparisonrevealed a significant difference between the Preext1-Vehiclegroup and the other groups (Ext1: Preext1-WIN: P 5 0.007;Preext1-AM404: P 5 0.007) (Ext2: Preext1-WIN: P 5 0.004;Preext1-AM404: P 5 0.003). Thus, the reduced latencies toenter the dark side of the box on Ext1 and Ext2 indicate that

WIN and AM404 in the CA1 impair IA retrieval and facilitateextinction. Although the extinction kinetics in the preext1-vehi-cle groups in Figures 2a,b does not seem to be the same, repeatedmeasures ANOVA [treatment 3 days (2 3 5)] did not reveal asignificant difference between the groups in terms of their latencyto enter the dark side of the box (F(1,18) < 1, NS), or a significantinteraction effect (F(1,18) 5 1.367, NS). The within-subjectlatencies differed significantly between the days (F(1,18) 529.831, P < 0.001), probably due to the increased latency onExt1 in the preext1-vehicle group in Figure 2b.

Next, we examined the effects of the agonists on IA condi-tioning. Repeated measures ANOVA [treatment 3 days (4 35)] revealed a significant difference between the groups in termsof their latency to enter the dark side of the box (F(2,20) 55.8,P 5 0.01; Fig. 2c), with no significant within-subject difference

in the latency between the days (F(1,20) < 1, NS) and no inter-action effect (F(2,20) 5 2.1, NS). Post-hoc comparison unveiled

a significant difference between rats microinjected with AM404before conditioning (Precond-AM404, n 5 7) and the groupsmicroinjected with WIN (Precond-WIN, n 5 8: P 5 0.008)or vehicle (Precond-Vehicle, n 5 8: P 50.006).

One-way ANOVA on the different days of the experimentrevealed that the significant main effect stemmed from a differ-ence in latency between the groups on Ext3 (F(2,20) 5 5.8,P 5 0.01), Ext4 (F(2,20) 5 3.47, P 5 0.051), and a strongtrend on Ext 2 (F(2,20) 5 2.99, P 5 0.073). Post-hoc compari-son revealed that the latency of the Precond-AM404 group was

significantly greater than that of the other groups (Ext2: Pre-cond-WIN: P 5 0.048; Precond-Vehicle: P 5 0.042) (Ext3:Precond-WIN: P 5 0.01; Precond-Vehicle: P 5 0.006) (Ext4:Precond-WIN: P 5 0.026; Precond-Vehicle: P 5 0.04). Notethat on Ext1, the AM404 group exhibited its maximal latencyto enter the dark side (i.e., 180 s), suggesting enhanced IA con-

ditioning. Microinjecting a higher dose of the CB1 receptoragonist WIN55,212-2 (10 lg/0.5 ll) before conditioning hadno significant effect on IA acquisition or extinction (data notshown). Thus, AM404 microinjected into the CA1 before con-ditioning facilitated IA acquisition, which probably resulted inimpaired extinction. WIN55,212-2 microinjected before condi-tioning had no effect on either IA conditioning or extinction.

The Effects of the Different Drugson Sensory-Motor Parameters

To exclude sensory-motor deficits, increased anxiety, andalterations to shock reactivity as causes for the observed drugeffects on IA conditioning and extinction, we performed severalcontrol experiments using open field, elevated plus-maze, andpain sensitivity tests.

Rats were microinjected into the CA1 with the CB1 receptorantagonist (AM251 6 ng, n 5 6), agonists (WIN 5 lg, n 5 6and AM404 200 ng, n 5 7), or vehicle (Vehicle, n 5 8) andthen tested in the open field arena, elevated plus-maze, andpain sensitivity tests. One-way ANOVA did not reveal a signifi-cant difference between the groups for any of the parametersmeasured in the open field test (Table 1), namely, time spent atthe center (F(3,23) < 1, NS ), time spent in the periphery

(F(3,23) < 1, NS), resting time (

F(3,23) < 1, NS), number ofrearing events (F(3,23) 5 2.38, NS), or the distance covered

(F(3,23) 5 1.489, NS).Similarly, one-way ANOVA did not reveal a significant dif-

ference between the groups in any of the parameters measuredin the elevated plus-maze test (Table 2), namely, the percentageof time spent in the open arms (F(3,23) < 1, NS), the percent-age of time spent in the closed arms (F(3,23) < 1, NS). Further,no significant differences between the groups were observed inthe percentage of entries into open arms (F(3,23) < 1, NS), thenumber of entries into the open (F(3,23) < 1, NS), or the num-ber of entries into the closed arms (F(3,23)


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In the pain sensitivity test, one-way ANOVA analysisrevealed marginally significant differences between the pain sen-sitivity thresholds of the treatment groups (F(3,23) 5 3.275,P 5 0.06). Post-hoc comparison unveiled that the AM404

group had a significantly lower pain sensitivity threshold thanthe vehicle group (P< 0.05) (Table 3).

The Effects of Cannabinoid Activation andDeactivation on the Induction of LTP inthe Schaffer Collateral-CA1 Pathway andSpatial Learning

To test the involvement of the CB1 receptor in the induc-tion of LTP, rats were i.p. injected with AM251 (1 mg/kg or2 mg/kg) 30 min before applying HFS to the Schaffer collat-eral. Repeated measures ANOVA [treatment 3 time (3 3 18)]post-HFS indicated significant effects on EPSP amplitude forthe treatment (F(2,15) 5 9.461, P 5 0.02), but not for thetime (F(1,15) < 1, NS) or the interaction between treatmentand time (F(2,15) < 1, NS; Fig. 3a). Post-hoc analysis revealeda significant reduction in EPSP amplitude in rats injected with

AM251 (AM251 2 mg, n 5 6 and AM251 1 mg, n 5 4),compared with the vehicle group (vehicle, n 5 8; P < 0.01).Hence, the cannabinoid antagonist AM251 impaired the induc-tion of LTP in the Schaffer collateral-CA1 pathway. Repeatedmeasures ANOVA on EPSP amplitude pre-HFS [treatment 3time (3 3 6)] did not reveal significant effects for the treat-ment (F(2,15) < 1, NS), the time (F(1,15) < 1, NS), or theinteraction between treatment and time (F(2,15)


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7)] of the acquisition day data revealed a significant differencebetween the groups in terms of their latencies to locate the hid-den platform (F(3,28) 5 6.05, P < 0.01; Fig. 3d). Also, therewas a significant within-subject difference in latency betweenthe trials (F(1,28) 5 28.096, P< 0.001) and a significant inter-

action between treatment and trials (F(3,28) 5 6.26, P< 0.01).A post-hoc comparison of the latency to locate the hidden plat-form indicated a significant difference between the vehiclegroup (n 5 9) and the treated groups (AM251, n 5 7: P

Cannabinoids Modulate Hippocampal Memory and Plasticity

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