Cannabinoids Modulate Hippocampal Memory and Plasticity

Hila Abush and Irit Akirav

*
ABSTRACT: Considerable evidence demonstrates that cannabinoid
agonists impair whereas cannabinoid antagonists improve memory and
plasticity. 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 investigated
the involvement of the cannabinoid system in hippocampal learning and
plasticity using the fear-related inhibitory avoidance (IA) and the non-
fear-related spatial learning paradigms, and cellular models of learning
and 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 the
extinction 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 in
pain sensitivity. However, in the water maze, systemic or local CA1
injections of AM251, WIN55,212-2, and AM404 all impaired spatial
learning. 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 in
the Schaffer collateral-CA1 projection, whereas AM404 facilitated LTD.
Our ndings suggest diverse effects of the cannabinoid system on CA1
memory 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 treatment
of disorders associated with impaired extinction-like processes, such as
post-traumatic stress disorder. V VC 2009 Wiley-Liss, Inc.
KEY WORDS: CB1 receptors; extinction; LTP; LTD; avoidance; CA1
INTRODUCTION
Learning and memory impairments are among the most commonly
reported behavioral effects of cannabinoids (Lichtman et al., 1995; Pam-
plona and Takahashi, 2006). These effects are thought to be associated
with the hippocampus, an area highly expressed with cannabinoid recep-
tors (Berrendero et al., 1999). Systemic administration of cannabinoid
agonists induces decits in several hippocampal dependent tasks, such as
the 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 delayed
alternation 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. On
the other hand, studies using CB1 receptor antago-
nists or CB1-decient mice usually demonstrate
increased memory performance in hippocampal-
dependent memory tasks like the radial maze
(Lichtman, 2000) and social recognition (Terranova
et al., 1996).
Similarly, previous studies indicate that cannabinoid
receptor activation in hippocampal slices impairs long-
term potentiation (LTP; Nowicky et al., 1987; Collins
et al., 1994; Terranova et al., 1995) and long-term
depression (LTD; Misner and Sullivan, 1999), whereas
CB1 blockade enhances LTP in hippocampal slices,
and a similar enhancement of LTP is observed in
mice lacking cannabinoid CB1 receptors (Bohme
et al., 2000; Slanina et al., 2005).
Taken together, considerable evidence demonstrates
that cannabinoid agonists impair whereas cannabinoid
antagonists improve memory and plasticity (for
review, Riedel and Davies, 2005). However, recent
studies suggest that the effects of cannabinoids on
learning do not necessarily follow these previously
described simple patterns, particularly when emotional
memory processes are involved (Chhatwal and Ressler,
2007; Lutz, 2007).
An increasing number of studies demonstrate that
the cannabinoid system is critical for the encoding of
emotional memory and for the extinction of fear-
related memories (Marsicano et al., 2002; Milad and
Quirk, 2002; Chhatwal et al., 2005; Laviolette and
Grace, 2006a,b; Viveros et al., 2007). CB1-decient
mice show impaired auditory fear extinction, with
unaffected memory acquisition and consolidation
(Marsicano et al., 2002). Additionally, there are
reports of facilitated fear extinction following systemic
cannabinoid activation (Chhatwal et al., 2005; Pam-
plona et al., 2006, 2008) although several reports
failed to show accelerated extinction following the
administration of cannabinoid agonists WIN55,212-2
and tetrahydrocannabinol (THC; Chhatwal et al.,
2005; Kobilo et al., 2007; Varvel et al., 2007).
Thus, in the present set of experiments, we aimed
to study whether emotional and nonemotional mem-
ory formation and two models of synaptic plasticity
are differentially modulated by the cannabinoid sys-
tem. Hence, we examined the effects of exogenously
administered cannabinoid receptor agonist and antag-
onist on a hippocampal-dependent fear-related mem-
ory paradigm [i.e., inhibitory avoidance (IA)] and a
non-fear-based paradigm (i.e., spatial learning). In
Department of Psychology, University of Haifa, Haifa, Israel
Grant sponsor: The National Institute for Psychobiology, Israel; Grant
number: 203-07-08 (to I.A.).
*Correspondence to: Irit Akirav, PhD, Department of Psychology, Univer-
sity of Haifa, Haifa 31905, Israel. E-mail: irit.akirav@gmail.com
Accepted for publication 20 August 2009
DOI 10.1002/hipo.20711
Published online 14 October 2009 in Wiley Online Library
(wileyonlinelibrary.com).
HIPPOCAMPUS 20:11261138 (2010)
V VC
2009 WILEY-LISS, INC.
addition, we examined the effects of these drugs on LTP and
its 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) were
caged individually at 22 6 28C under 12-h light/dark cycles
(lights turned on at 07:00 and turned off at 19:00). Rats had
access to water and laboratory rodent chow ad libitum. The
experiments were approved by the University of Haifa Ethics
and Animal Care Committee, and adequate measures were
taken to minimize pain or discomfort in accordance with the
guidelines laid down by the National Institutes of Health in
the US regarding the care and use of animals for experimental
procedures.
Except for the sensory-motor sequence tests, every rat under-
went one behavioral test or electrophysiological test, with the
different drugs (or drug doses) to prevent carryover effects due
to 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). Final
DMSO concentration was <7%. This DMSO and saline solu-
tion was also used as the vehicle.
The nal DMSO concentration did not affect performance
in the IA or spatial tasks, nor did it affect basal synaptic trans-
mission or LTP. Drug concentrations are based on reports in
the literature (Martin et al., 1999; Chhatwal et al., 2005; De
Oliveira Alvares et al., 2005; Pamplona et al., 2006; Moreira
et al., 2007) and our previous results (Ganon-Elazar and
Akirav, 2009).
For microinjection: WIN 55,212-2: 5 lg/0.5 ll or 10 lg/
0.5 ll, AM404: 200 ng/0.5 ll, AM251: 6 ng/0.5 ll. For intra-
peritoneal (i.p.) administration: WIN 55,212-2: 0.25 or
0.5 mg/kg, AM404: 5 or 10 mg/kg, AM251: 1 or 2 mg/kg.
Drugs were administered at a volume of 1 ml/kg of body
weight.
Cannulation and Drug Microinjection
Rats were anesthetized with 4.8 ml/kg Equithesin (2.12% w/v
MgSO
4
, 10% ethanol, 39.1% v/v propylene glycol, 0.98% w/v
sodium pentobarbital, and 4.2% w/v chloral hydrate), restrained
in a stereotactic apparatus (Stoelting, Wood Dale, IL) and
implanted bilaterally with a stainless steel guide cannula (23-
gauge, thin wall) aimed at the dorsal CA1 (anteroposterior, 24.2
mm; lateral, 62.5 mm; ventral, 22.3 mm). The cannulae were
set in place with acrylic dental cement and secured by two skull
screws. A stylus was placed in the guide cannula to prevent clog-
ging. Animals were allowed 1 week to recuperate before being
subjected to experimental manipulations.
For microinjection, the stylus was removed from the guide
cannula and a 28-gauge injection cannula, extending 1.0 mm
from the tip of the guide cannula, was inserted. The injection
cannula was connected via PE20 tubing to a Hamilton micro-
syringe driven by a microinfusion pump (CMA/100; Carnegie
Medicine, Stockholm, Sweden). A volume of 0.5 ll per side
was microinjected bilaterally over a period of 1 min. The injec-
tion cannula was left in position for an additional 30 s before
withdrawal to minimize dragging of the injected liquid along
the injection tract.
LightDark Inhibitory Avoidance
Animals were individually placed in a shuttle box with a
metal grid oor. The box was divided into a light and a dark
side, and the rat was placed in the light side, facing the left
rear corner of the box. The shuttle box itself was placed in a
dimly lit room (Tinsley et al., 2004).
For conditioning (Cond), when the rat crossed over to the
dark side of the box (with four paws on the grid), it received a
2 s, 0.7 mA scrambled footshock. Following administration of
the footshock, the opening between the two sides of the box
was blocked and the rat remained in the dark side for an addi-
tional 60 s, after which it was removed back to the home cage.
For extinction, rats were submitted to a nonreinforced test
trial every 24 h for 3 days (Ext1-Ext3), beginning 24 h post-
conditioning. Each rat was put back in the light side of the
box until it crossed over to the dark side. If the rat did not
cross over within 180 s, the experimenter gently guided it to
the dark side. The opening between the two sides of the box
was then blocked, no footshock was administered, and the rat
was allowed to explore the dark side freely for 180 s, after
which it was removed back to the home cage.
To examine effects on IA conditioning and extinction, drugs
were microinjected into the CA1 20 min before conditioning
(Pre-Cond) or 20 min before the rst extinction trial (pre-
Ext1). Control rats received microinjections of the vehicle at
the same time points.
Histology
Upon completion of the behavioral experiments, the animals
were deeply anesthetized, and 0.5 ll of Indian ink was micro-
injected into the CA1 to verify the location of the cannulae.
Figure 1a shows a schematic drawing of the placements of the
cannulae in the CA1 (coronal view at position 3.8 and 4.16
mm anterior to bregma) (Paxinos and Watson, 1998). Solid
black circles indicate the locations in a subset of animals (not
all animals are shown in light of the number of rats involved
in the experiments). The indicated numbers of rats per each
group are the nal group sizes after controlling the location of
the cannulae by histology.
CANNABINOIDS FACILITATE EXTINCTION AND IMPAIR LTP 1127
Hippocampus
Another set of rats implanted bilaterally with cannula into
the CA1 were anesthetized and perfused transcardially with
200 ml of saline, followed by 200 ml of 4% paraformaldehyde
(PFA), pH 7.4. The brains were removed and then immersed
in 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. Slides
were examined under a light microscope. Figure 1b shows pho-
tomicrograph illustrating a typical cannula track in CA1 of
representative brain section. As we used a small volume of
infusion (0.5 ll), the possibility of injection spread to other
hippocampal subelds is little.
Sensory-Motor Tests
Three tests to investigate sensory-motor parameters were pre-
sented to the rats in sequence: an open eld, an elevated plus-
maze, and a pain sensitivity tests (see below). All tests were
conducted in dimly lit rooms. Drugs were microinjected into
the 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 rst experienced the open eld test, while the
other half began with the elevated plus-maze. The pain sensitiv-
ity test was always third in the sequence, because it involved a
footshock that could otherwise have affected performance in
the open eld and maze tasks. The open eld and elevated plus
maze tests were conducted for 5 min each, with 5-min acclima-
tion to the test chamber prior to testing.
Open eld
The open eld consisted of a closed wooden box. The walls
were painted black and the oor was white and divided by 1-
cm-wide black lines into 25 squares measuring 10 3 10 cm
2
each. A video image of the entire open eld was displayed on a
TV monitor, and the movements of the rat, which was initially
placed in a corner of the eld, 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 central
and the peripheral squares, the number of instances of rearing,
and the total distance covered. The open eld 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 oor of the maze was white. Two
of the arms had no railing (open arms), and the other two
arms were protected by opaque railings (painted black) to a
height 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 spent
in each of the different arms, and the number of entries into
the closed and open arms were manually recorded for a period
of 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., inch or
vocalization) (Kim et al., 1991). Rats were individually placed
in a Plexiglas box (25 3 25 3 34 cm
3
) with a oor consisting
of 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 animal
showed discomfort) via the metal grid oor to determine cur-
rent thresholds at which each animal would exhibit a inch or
a vocalization response. Two observers independently scored
FIGURE 1. Schematic drawing of cannulae tip positions in the
CA1 area of the hippocampus. (a) Schematic drawings of CA1 can-
nulae placements. Shown is a coronal view at position 4.16 and
4.3 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 4.16
mm posterior to bregma). The full arrow represents the position of
the tip of the cannula and the dashed arrow illustrates where the
position of the injection cannula would be (extending 1 mm
beyond the tip of the cannula into the CA1).
1128 ABUSH AND AKIRAV
Hippocampus
inch and vocalization thresholds. We found that all the rats
showed a inch response that was sometimes accompanied by a
vocalization response. Thus, the inch response determined the
threshold.
Electrophysiology
Surgical procedure
Rats were anesthetized (with 40% urethane, 5% chloral
hydrate in saline, injection volume of 4 ml/1 kg, i.p.) and
placed in a stereotaxic frame. Small burr holes were drilled in
the skull to allow electrodes to be inserted into the brain. A re-
cording microelectrode (glass, tip diameter of 25 lm, lled
with 2 M NaCl, resistance of 14 M) was slowly lowered into
the CA1 area (anteroposterior, 24.2 mm; lateral, 62.5 mm;
ventral, 22 to 3 mm). A bipolar 125-lm stimulating electrode
was positioned to activate the Schaffer collateral-CA1 projec-
tion (anteroposterior, 23.1 mm; lateral, 60.4 mm; ventral,
23.5 mm). After positioning the electrodes, the rat was left for
60 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; each
train consisting of 10 pulses at 200 Hz; intertrain interval,
200 ls; interset interval, 1 min). Field potentials were recorded
from the CA1 every 5 min for 90 min after HFS to the
Schaffer 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 the
Schaffer collateral (4 Hz). Field potentials were recorded from
the 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. Ofine
measurements were made of EPSP amplitude using averages of
ve successive responses to a given stimulation intensity applied
at 0.1 Hz (Akirav and Richter-Levin, 2002).
Drugs were i.p. injected 20 min (vehicle, WIN55,212-2, and
AM404) or 30 min (AM251, see Sink et al., 2008) before
applying HFS or LFS to the Schaffer collateral.
The Morris water maze task
The maze was placed in a dimly lit room. Animals were
individually placed at several start locations around the maze
and were required to nd the location of a hidden platform
within 60 s. If a rat did not reach the platform within 60 s, an
experimenter would guide it there (Roozendaal et al., 2004).
The rat was then allowed to remain on the platform for 25 s
before removal back to the home cage, to enable the rat to
learn 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 were
tested for their ability to locate the escape platform (1-min
trial). Drugs were either injected i.p. or microinjected into the
CA1, 30 min before the rst acquisition trial.
Statistical Analysis
The results are expressed as means 6 SEM. For statistical
analysis, repeated-measures ANOVA for treatment 3 days (IA),
treatment 3 blocks (spatial learning), or treatment 3 time
(electrophysiology) was used. One-way ANOVA was used to
compare between the different drug treatments. t-test was used
in the water maze task. All post-hoc comparisons were made
using the least signicant difference multiple-comparison test
(LSD).
Experimental Design
Inhibitory avoidance
Eighty-ve rats were implanted with cannulae into the CA1
and 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 rst extinction trial (pre-Ext1). Each rat
was microinjected once.
Electrophysiology
A second group of rats (n 5 81) was anesthetized and taken
for electrophysiological recording in the CA1. Drugs or vehicle
were i.p. injected 2030 min prior to HFS or LFS. In another
set of animals (n 5 20), drugs or vehicle were i.p. injected
with no HFS or LFS (Fig. 4b).
Spatial learning
Thirty-two rats were implanted with cannulae into the CA1
and left 1 week to recuperate. Drugs or vehicle were microin-
jected into the CA1 30 min before spatial training in the water
maze. In another set of rats (n 5 34), drugs or vehicle were
i.p. injected 30 min before spatial training in the water maze.
Sensory-motor parameters
Twenty-seven rats were implanted with cannulae into the
CA1 and left 1 week to recuperate. Drugs or vehicle were
microinjected into the CA1 20 min before the rst sensory-
motor test (the open eld or the elevated plus-maze).
CANNABINOIDS FACILITATE EXTINCTION AND IMPAIR LTP 1129
Hippocampus
RESULTS
The Effects of Cannabinoid Activation and
Deactivation in the CA1 on Inhibitory
Avoidance Conditioning and Extinction
To examine whether the CB1 receptor in the CA1 is essen-
tial in IA conditioning and extinction, rats were microinjected
with the CB1 receptor antagonist AM251 (6 ng/0.5 ll) either
before conditioning (Cond) or before the rst extinction trial
(Ext1). Repeated measures ANOVA [treatment 3 days (4 3
5)] revealed a signicant difference between the groups in terms
of 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 not
differ signicantly between the days (F
(1,29)
5 2.57, NS), but
there was a signicant interaction effect (F
(3,29)
5 5.26, P 5
0.005).
Post-hoc comparison unveiled a signicant difference
between the group microinjected with AM251 prior to Ext1
(Preext1-AM251, n 5 8) and all the other groups, i.e., the
vehicle 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 5
0.006). One-way ANOVA on the different days of the experi-
ment revealed that the signicant main effect stemmed from a
difference 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 signicantly greater than that of the other
groups (Ext3: Precond-Vehicle: P 5 0.005; Preext1-Vehicle: P
5 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 acquisition
when microinjected before conditioning, but impaired extinc-
tion when microinjected before the rst extinction trial.
Following the clear result we obtained from using the CB1
receptor antagonist to block IA extinction, we next asked
whether stimulation of cannabinoid receptor signaling might
accelerate the extinction rate. A previous report (Chhatwal
et 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 rst extinc-
tion trial (Ext1). Repeated measures ANOVA [treatment 3
days (4 3 5)] revealed a signicant difference between the
groups in terms of their latencies to enter the dark side of the
FIGURE 2. The effects of cannabinoid receptor antagonist and
agonists into the CA1 on inhibitory avoidance conditioning and
extinction. (a) AM251 (6 ng/0.5 ll) microinjected into the CA1
before the rst extinction trial (Preext1-AM251, n 5 8) signi-
cantly increased the latency of the rats to enter the dark side of
the box compared with the vehicle groups (Preext1-Vehicle, n 5
10; Precond-Vehicle, n 5 8) and with the group that received a
microinjection 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 CA1
before the rst extinction trial (Preext1-WIN, n 5 9; Preext1-
AM404, n 5 8) signicantly decreased the latency of the rats to
enter the dark side of the box compared with the vehicle group
(Preext1-Vehicle, n 5 12) on Ext1 and Ext2, indicating facilitation
of 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) signicantly increased the la-
tency of the rats to enter the dark side of the box compared with
the 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).
1130 ABUSH AND AKIRAV
Hippocampus
box (F
(2,26)
5 5.92, P 5 0.008; Fig. 2b). The within-subject
latencies also differed signicantly between the days (F
(1,26)
5
3.92, P 5 0.051), but there was no interaction effect (F
(2,26)
5 1.44, NS). Post-hoc comparison unveiled a signicant differ-
ence between the vehicle group (Preext1-Vehicle, n 5 12) and
the groups microinjected with WIN (Preext1-WIN, n 5 9:
P 5 0.007) or AM404 (Preext1-AM404, n 5 8: P 5 0.009).
One-way ANOVA on the different experimental days revealed
that the signicant 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 comparison
revealed a signicant difference between the Preext1-Vehicle
group 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 to
enter the dark side of the box on Ext1 and Ext2 indicate that
WIN and AM404 in the CA1 impair IA retrieval and facilitate
extinction. Although the extinction kinetics in the preext1-vehi-
cle groups in Figures 2a,b does not seem to be the same, repeated
measures ANOVA [treatment 3 days (2 3 5)] did not reveal a
signicant difference between the groups in terms of their latency
to enter the dark side of the box (F
(1,18)
< 1, NS), or a signicant
interaction effect (F
(1,18)
5 1.367, NS). The within-subject
latencies differed signicantly between the days (F
(1,18)
5
29.831, P < 0.001), probably due to the increased latency on
Ext1 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 3
5)] revealed a signicant difference between the groups in terms
of their latency to enter the dark side of the box (F
(2,20)
5 5.8,
P 5 0.01; Fig. 2c), with no signicant 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 signicant difference between rats microinjected with AM404
before conditioning (Precond-AM404, n 5 7) and the groups
microinjected with WIN (Precond-WIN, n 5 8: P 5 0.008)
or vehicle (Precond-Vehicle, n 5 8: P 5 0.006).
One-way ANOVA on the different days of the experiment
revealed that the signicant 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 strong
trend 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
signicantly 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). Note
that on Ext1, the AM404 group exhibited its maximal latency
to enter the dark side (i.e., 180 s), suggesting enhanced IA con-
ditioning. Microinjecting a higher dose of the CB1 receptor
agonist WIN55,212-2 (10 lg/0.5 ll) before conditioning had
no signicant effect on IA acquisition or extinction (data not
shown). Thus, AM404 microinjected into the CA1 before con-
ditioning facilitated IA acquisition, which probably resulted in
impaired extinction. WIN55,212-2 microinjected before condi-
tioning had no effect on either IA conditioning or extinction.
The Effects of the Different Drugs
on Sensory-Motor Parameters
To exclude sensory-motor decits, increased anxiety, and
alterations to shock reactivity as causes for the observed drug
effects on IA conditioning and extinction, we performed several
control experiments using open eld, elevated plus-maze, and
pain sensitivity tests.
Rats were microinjected into the CA1 with the CB1 receptor
antagonist (AM251 6 ng, n 5 6), agonists (WIN 5 lg, n 5 6
and AM404 200 ng, n 5 7), or vehicle (Vehicle, n 5 8) and
then tested in the open eld arena, elevated plus-maze, and
pain sensitivity tests. One-way ANOVA did not reveal a signi-
cant difference between the groups for any of the parameters
measured in the open eld test (Table 1), namely, time spent at
the center (F
(3,23)
< 1, NS ), time spent in the periphery
(F
(3,23)
< 1, NS), resting time (F
(3,23)
< 1, NS), number of
rearing 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 signicant dif-
ference between the groups in any of the parameters measured
in the elevated plus-maze test (Table 2), namely, the percentage
of 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 signicant differences between the groups were observed in
the percentage of entries into open arms (F
(3,23)
< 1, NS), the
number of entries into the open (F
(3,23)
< 1, NS), or the num-
ber of entries into the closed arms (F
(3,23)
< 1, NS).
TABLE 1.
The Effects of Cannabinoid Receptor Antagonist and Agonists Microinjected Into the CA1 on Locomotion and Anxiety in the Open Field Test
Group Time at the center (s) Time at the periphery (s) Resting time (s) Number of rearings Distance covered (cm)
Vehicle, n 5 8 10.88 6 2.73 289.13 6 2.73 73.75 6 13.66 23 6 2.17 1316.25 6 84.39
AM404 200 ng, n 5 7 9.71 6 3.1 290.29 6 3.1 74.43 6 8.93 29.14 6 2.22 1391.43 6 160.36
WIN 5 lg, n 5 6 5.5 6 2.2 294.67 6 2.2 80 6 4.93 24.5 6 1.52 1395 6 46.82
AM251 6 ng, n 5 6 9 6 1.34 291 6 1.34 69.33 6 13.75 21.83 6 2.15 1625 6 90.32
The behavior of rats microinjected into the CA1 with the CB1 receptor antagonist (AM251 6 ng, n 5 6) or agonists (WIN 5 lg, n 5 6; AM404 200 ng, n 5 7)
or vehicle (Vehicle, n 5 8) did not differ in terms of any of the parameters measured in the open eld test.
CANNABINOIDS FACILITATE EXTINCTION AND IMPAIR LTP 1131
Hippocampus
In the pain sensitivity test, one-way ANOVA analysis
revealed marginally signicant 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 signicantly lower pain sensitivity threshold than
the vehicle group (P < 0.05) (Table 3).
The Effects of Cannabinoid Activation and
Deactivation on the Induction of LTP in
the Schaffer Collateral-CA1 Pathway and
Spatial 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 or
2 mg/kg) 30 min before applying HFS to the Schaffer collat-
eral. Repeated measures ANOVA [treatment 3 time (3 3 18)]
post-HFS indicated signicant effects on EPSP amplitude for
the treatment (F
(2,15)
5 9.461, P 5 0.02), but not for the
time (F
(1,15)
< 1, NS) or the interaction between treatment
and time (F
(2,15)
< 1, NS; Fig. 3a). Post-hoc analysis revealed
a signicant 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. Repeated
measures ANOVA on EPSP amplitude pre-HFS [treatment 3
time (3 3 6)] did not reveal signicant effects for the treat-
ment (F
(2,15)
< 1, NS), the time (F
(1,15)
< 1, NS), or the
interaction between treatment and time (F
(2,15)
< 1, NS).
To test the effects of CB1 receptor agonist on the induction
of LTP, another set of rats was i.p. injected with WIN55,212-2
20 min before applying HFS to the Schaffer collateral.
Repeated measures ANOVA [treatment 3 time (3 3 18)]
post-HFS indicated signicant effects on EPSP amplitude for
the treatment (F
(2,16)
5 5.549, P 5 0.015), but not for the
time (F
(1,16)
< 1, NS) or the interaction between treatment
and time (F
(2,16)
< 1, NS; Fig. 3b). Post-hoc analysis revealed
signicantly reduced EPSP amplitudes in rats injected with the
higher dose of WIN55,212-2 (WIN 0.5 mg, n 5 6) compared
with the vehicle group (vehicle, n 5 8; P 5 0.005), and a
strong trend between rats injected with the lower dose of
WIN55,212-2 (WIN 0.25 mg, n 5 5) and the vehicle group
(P 5 0.079). Hence, the cannabinoid receptor agonist
WIN55,212-2 impaired the induction of LTP in the Schaffer
collateral-CA1 pathway in a dose-dependent manner.
Repeated measures ANOVA on EPSP amplitude pre-HFS
[treatment 3 time (3 3 6)] did not reveal signicant effects
for the treatment (F
(2,16)
5 1.651, NS), the time (F
(1,16)
< 1,
NS), or the interaction between treatment and time (F
(2,16)
<
1, NS). Representative traces in the CA1 for the vehicle and
the WIN0.5 mg/kg groups taken before and after HFS to the
Schaffer collateral are shown as an inset in Figure 3b. Another
set of rats was i.p. injected with AM404 (5 mg/kg or 10 mg/
kg) 20 min before applying HFS to the Schaffer collateral.
Repeated measures ANOVA [treatment 3 time (3 3 18)] on
the post-HFS data indicated signicant effects on EPSP ampli-
tude for the treatment (F
(2,13)
5 5.866, P 5 0.015), but not
for the time (F
(1,13)
< 1, NS) or the interaction between treat-
ment and time (F
(1,13)
5 2.819, NS; Fig. 3c). Post-hoc analysis
revealed signicantly reduced EPSP amplitudes in rats injected
with the higher (AM404 10 mg, n 5 6; P 5 0.007) and lower
(AM404 5 mg, n 5 4; P < 0.042) doses of AM404 compared
with the vehicle group (vehicle, n 5 7). Hence, the endocanna-
binoid reuptake and breakdown inhibitor AM404 also
impaired the induction of LTP in the Schaffer collateral-CA1
pathway. Repeated measures ANOVA on the pre-HFS data
[treatment 3 time (3 3 6)] did not reveal signicant effects
for the treatment (F
(2,13)
5 3.548, NS), the time (F
(1,13)
< 1,
NS), or the interaction between treatment and time (F
(2,13)
5
2.585, NS).
Since both the antagonist (AM251) and the agonists
(AM404 and WIN) impaired hippocampal LTP, we sought to
examine whether cannabinoid activation and deactivation
would also impair behavioral encoding of another hippocam-
pal-dependent learning paradigm that is non-fear-related. Thus,
we used systemic and local injections to examine the effects of
the cannabinoids receptor agonists and antagonist on the acqui-
sition of a spatial task in the Morris water maze.
First, we examined the effects of AM251 (6 ng/0.5 ll),
WIN55,212-2 (5 lg/0.5 ll), AM404 (200 ng/0.5 ll), or vehi-
cle microinjected into the CA1 on the acquisition of the spatial
task. Repeated measures ANOVA [treatment 3 blocks (4 3
TABLE 2.
The Effects of Cannabinoid Receptor Antagonist and Agonists Microinjected Into the CA1 on Locomotion and Anxiety
in the Elevated Plus-Maze Test
Group
Time in open
arms (%)
Time in closed
arms (%)
Entries into
open arms (%)
Number of entries
into open arms
Number of
entries into
closed arms
Vehicle, n 5 8 30.75 6 8.5 69.25 6 8.5 38.92 6 8.24 4.75 6 1.69 4.88 6 0.58
AM404 200 ng, n 5 7 22.54 6 7.07 77.46 6 7.07 41.87 6 9.63 3.57 6 0.92 3.86 6 1.03
WIN 5 lg, n 5 6 14.93 6 5.13 85.07 6 5.13 32.64 6 10.99 2.83 6 1.01 3.67 6 0.76
AM251 6 ng, n 5 6 19.07 6 8.44 80.93 6 8.44 31.25 6 10.08 2.5 6 1.06 3.67 6 0.88
The behavior of rats microinjected into the CA1 with the CB1 receptor antagonist (AM251 6 ng, n 5 6) or agonists (WIN 5 lg, n 5 6; AM404 200 ng, n 5 7)
or vehicle (Vehicle, n 5 8) did not differ in terms of any of the parameters measured in the elevated plus-maze test.
1132 ABUSH AND AKIRAV
Hippocampus
7)] of the acquisition day data revealed a signicant difference
between 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, there
was a signicant within-subject difference in latency between
the trials (F
(1,28)
5 28.096, P < 0.001) and a signicant 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 signicant difference between the vehicle
group (n 5 9) and the treated groups (AM251, n 5 7: P <
0.001; WIN, n 5 8: P < 0.05; AM404, n 5 8: P 5 0.052),
and between the AM251 group and the other groups (WIN:
P < 0.05; AM404: P < 0.05).
Twenty-four hours after training, rats were tested in the
maze. One-way ANOVA revealed a signicant difference
between the groups in terms of their latency to locate the hid-
den platform (F
(3,28)
5 3.8, P < 0.05). Post-hoc comparison
indicated a signicant difference between the vehicle group and
the AM251 (P < 0.05) and AM404 (P 5 0.003) groups.
Thus, cannabinoid antagonist and agonists microinjected into
the CA1 before training impaired spatial learning in the water
maze. Note that in this short intertrial interval training proto-
col, control rats show relatively increased latency in the retrieval
test compared to control rats trained in a training protocol
with a longer intertrial interval (Akirav et al., 2001).
Next, we examined the effects of AM251 (1 mg/kg),
WIN55,212-2 (0.5 mg/kg), AM404 (5 mg/kg), or vehicle
administered i.p. on the acquisition of the spatial task.
Repeated measures ANOVA [treatment 3 blocks (4 3 7)] of
the acquisition day data revealed a marginally signicant differ-
ence between the groups in terms of their latencies to locate
the hidden platform (F
(3,30)
5 2.786, P 5 0.058; Fig. 3e).
Also, there was a signicant within-subject difference in latency
between the trials (F
(1,30)
5 174.672, P < 0.001). A post-hoc
comparison of the latency to locate the hidden platform indi-
cated a signicant difference between the vehicle group (n 5
11) and the AM251 and the AM404 groups (P < 0.05; n 5 7
each). An independent samples t-test of the last block of
training revealed a signicant difference between the vehicle
group and the WIN-treated group (n 5 9; t
(18)
5 22.295,
P < 0.05).
Twenty-four hours after training, rats were tested in the
maze. One-way ANOVA revealed a signicant difference
between the groups in terms of their latency to locate the hid-
den platform (F
(3,30)
5 5.119, P 5 0.006). Post-hoc compari-
son indicated a signicant difference between the AM404
group and the other groups (Vehicle: P < 0.001, WIN: P <
0.05, AM251: P < 0.05). An independent samples t-test of the
test day revealed a signicant difference between the vehicle
group and the WIN-treated group (t
(18)
5 22.907, P < 0.01),
as well as between the vehicle group and the AM251-treated
group (t
(16)
5 22.113, P 5 0.051). Thus, cannabinoid antag-
onist and agonists injected i.p. before training impaired spatial
performance in the water maze.
The Effects of Cannabinoid Activation and
Deactivation on the Induction of LTD in the
Schaffer Collateral-CA1 Pathway
To test the involvement of the CB1 receptor in the induc-
tion of LTD, rats were i.p. injected with AM251 (2 mg/kg),
WIN55,212-2 (0.5 mg/kg), AM404 (10 mg/kg) or vehicle 20
or 30 minutes before applying LFS to the Schaffer collateral.
We selected these doses because we had found them effective in
impairing LTP (see above). Repeated measures ANOVA [treat-
ment 3 time (4 3 18)] post-LFS indicated signicant effects
on EPSP amplitude for treatment (F
(3,25)
5 3.258, P 5 0.038)
and time (F
(1,25)
5 4.286, P 5 0.049), but not for the interac-
tion between treatment and time (F
(3,25)
5 1.379, NS; Fig.
4a). Post-hoc analysis revealed a signicant enhancement of
LTD in rats injected with AM404 (AM404 10 mg, n 5 5)
compared with the other groups (Vehicle, n 5 9, P 5 0.029;
WIN 0.5 mg, n 5 8, P < 0.01; AM251 2 mg, n 5 6, P 5
0.015). Repeated measures ANOVA on EPSP amplitude pre-
LFS [treatment 3 time (4 3 6)] did not reveal signicant
effects for the treatment (F
(3,25)
< 1, NS), the time (F
(1,25)
<
1, NS), or the interaction between treatment and time (F
(3,25)
5 1.072, NS). Representative traces in the CA1 for the vehicle
and AM404 groups taken before and after LFS to the Schaffer
collateral are shown as an inset in Figure 4a.
Because of the variability in pre-tetanus levels, we included a
control experiment in which rats received only 0.1 Hz test
stimulation over the same duration of time as the HFS/LFS
groups. Rats were i.p. injected with the different drugs (Vehi-
cle, AM251 2 mg, WIN 0.5 mg, AM404 10 mg; n 5 5 each)
and no HFS or LFS were applied. Repeated measures ANOVA
[treatment 3 time (4 3 20)] indicated no signicant effects on
EPSP amplitude for the treatment (F
(3,16)
< 1, NS), the time
(F
(1,16)
5 4.024, NS), or the interaction between treatment
and time (F
(3,16)
< 1, NS) (Fig. 4b). Hence, the drugs did not
signicantly affect basal synaptic transmission.
DISCUSSION
Our ndings suggest that the neural processes underlying
memory formation in the dorsal hippocampus are differentially
sensitive to cannabinoid receptor activation and deactivation
depending on the type of memory under examination. We
TABLE 3.
The Effects of Cannabinoid Receptor Antagonist and Agonists
Microinjected Into the CA1 on Pain Sensitivity
Group Pain threshold (mA)
Vehicle, n 5 8 0.23 6 0.03
AM404 200 ng, n 5 7 0.14 6 0.02*
WIN 5 lg, n 5 6 0.22 6 0.02
AM251 6 ng, n 5 6 0.21 6 0.02
The pain sensitivity threshold of rats microinjected into the CA1 with AM404
(AM404 200 ng, n 5 7) was signicantly lower than that of the vehicle group
(Vehicle, n 5 8; *P < 0.05).
CANNABINOIDS FACILITATE EXTINCTION AND IMPAIR LTP 1133
Hippocampus
found that the cannabinoid receptor antagonist AM251 blocks
IA extinction, impairs spatial learning in the water maze, and
impairs LTP induction in the Schaffer collateral-CA1 pathway,
while having no effect on IA conditioning or LTD. Addition-
ally, the cannabinoid agonist WIN55,212-2 facilitates IA
extinction and impairs both spatial learning and LTP, while
having no effect on IA conditioning or LTD. Finally, AM404,
an inhibitor of endocannabinoid reuptake and breakdown,
facilitates IA conditioning and extinction, impairs spatial learn-
ing and LTP, and facilitates LTD.
FIGURE 3. CB1 receptor antagonist and agonists impair the
induction of LTP and performance in a spatial task. (a) AM251in-
jected i.p. (1 mg/kg, n 5 4 or 2 mg/kg, n 5 6) 30 min before applica-
tion of high frequency stimulation (HFS) to the Schaffer collateral sig-
nicantly impaired the induction of LTP in the CA1 compared with
the vehicle group (n 5 8) (P < 0.01, vehicle differs from all the
groups). (b) WIN55,212-2 (0.5 mg/kg, n 5 6) injected i.p. 20 min
before application of HFS to the Schaffer collateral signicantly
impaired the induction of LTP in the CA1 compared with the vehicle
group (n 5 8) (P 5 0.01). Inset: representative traces in the CA1 for
vehicle (upper traces) and WIN 0.5 mg (lower traces) groups taken
before (black) and 90 min after (gray) HFS to the Schaffer collateral
(calibration: 0.2 mV, 10 ls). (c) AM404 injected i.p. (5 mg/kg, n 5 4
or 10 mg/kg, n 5 6) 20 min before application of HFS to the Schaffer
collateral signicantly impaired the induction of LTP in the CA1 com-
pared with the vehicle group (n 5 7) (**P < 0.01, vehicle differs from
the higher dose group; *P < 0.05, vehicle differs from the lower dose
group). (d) AM251 (6 ng/0.5 ll, n 5 7), AM404 (200 ng/0.5 ll, n 5
8), or WIN55,212-2 (WIN 5 lg/0.5 ll, n 5 8) microinjected to the
CA1 before massed training in the Morris water maze signicantly
increased the latency of the rats to locate the hidden platform, com-
pared with the vehicle group (n 5 9) (*P < 0.05, vehicle differs from
the WIN and AM404 groups, ***P < 0.001, vehicle differs from the
AM251 group). Twenty-four hours post-training, the AM251 and
AM404 groups showed signicantly increased latencies to locate the
hidden platform compared with the vehicle group (*P < 0.05, vehicle
differs from AM251 group, **P < 0.01, vehicle differs from AM404
group). (e) AM251 (1 mg/1 kg, n 57), AM404 (5 mg/1 kg, n 57), or
WIN55,212-2 (0.5 mg/1 kg, n 5 9) injected i.p. before massed train-
ing in the Morris water maze signicantly increased the latency of the
rats to locate the hidden platform, compared with the vehicle group (n
5 11) (*P < 0.05). Twenty-four hours post-training, the WIN55,212-
2, AM404, and AM251 groups showed signicantly increased latencies
to locate the hidden platform compared with the vehicle group (*P <
0.05, vehicle differs from AM251 group; **P < 0.01, vehicle differs
from WIN group; ***P < 0.001, vehicle differs from AM404 group).
1134 ABUSH AND AKIRAV
Hippocampus
Animal studies using THC, synthetic cannabinoids, and
anandamide show that these substances cause hippocampal-
dependent memory decits (for a review, see Hampson and
Deadwyler, 1999). On the other hand, results by Marsicano
et al. (2002) and others have shown that cannabinoids mediate
fear extinction. Our results corroborate with these studies by
showing hippocampal-dependent memory impairment follow-
ing cannabinoid activation and cannabinoid mediation of the
extinction of inhibitory fear.
Yet, when taken together, our results indicate that the effects of
cannabinoids on learning and plasticity do not necessarily follow
the simple previously described patterns, i.e., that cannabinoid
agonists impair, whereas cannabinoid antagonists facilitate mem-
ory and plasticity. In general, the results lend support to the
recently made argument that cannabinoid agonists facilitate emo-
tional memory or extinction, and impair nonemotional memory,
but even this may underestimate the complexity of how cannabi-
noids modulate memory processes (for review, Riedel and Davies,
2005; Chhatwal and Ressler, 2007; Lutz, 2007).
The Involvement of the Cannabinoid
System in Inhibitory Avoidance
Conditioning and Extinction
Our ndings suggest that the CB1 receptor in the dorsal
hippocampus is crucial for the extinction of IA, since its antag-
onist AM251 had a long-term impairing effect on extinction
when microinjected into the CA1 area before extinction. Yet,
when microinjected before conditioning, AM251 in the dose
tested here (6 ng/0.5 ll) had no effect on IA conditioning or
on extinction kinetics, suggesting that its long-lasting blockade
of extinction is not the result of permanent tissue damage. Sim-
ilarly, AM251 had no effect on the acquisition of a step-down
IA task but blocked extinction of contextual fear conditioning
memory when microinfused into the CA1 (De Oliveira Alvares
et al., 2008a,b). This is in accordance with other studies that
have reported decits in extinction expression following sys-
temic administration of a CB1 receptor antagonist with no
effect on memory acquisition (Marsicano et al., 2002; Suzuki
et al., 2004; Chhatwal et al., 2005).
Conversely, cannabinoid receptor activation in the dorsal hip-
pocampus facilitated extinction. This facilitatory effect was evi-
dent in the rst two extinction days, and absent in the last two
extinction days, probably due to a oor effect. The reduced la-
tency to cross to the dark side in the rst extinction trial may sug-
gest that the facilitating effect of the cannabinoid agonists on
extinction is associated with the alleviation of fear. Nevertheless,
this is probably not due to a state-dependent effect, as facilitation
of extinction persisted in the following extinction trial (i.e.,
Ext2), which took place 24-h postdrug administration.
When microinjected before conditioning, WIN55,212-2 in
the dose tested here (5 lg/0.5 ll) had no signicant effect on
IA acquisition, further suggesting that CB1 receptors in the
CA1 are not involved in the conditioning of this task. How-
ever, AM404 signicantly facilitated IA conditioning and con-
sequently impaired extinction. Since a signicantly reduced
pain sensitivity threshold was observed following AM404 intra-
hippocampal microinjection, the enhanced latency observed fol-
lowing AM404 administration may result from increased sensi-
tivity to electric shock. Except for this effect of AM404 on
pain sensitivity, AM251, WIN55,212-2, and AM404 had no
effect on sensory-motor abilities, anxiety levels, or pain thresh-
old in control experiments. Therefore, the effects of the drugs
cannot be attributed to changes in these parameters.
The Involvement of the Cannabinoid System in
Hippocampal LTP and Spatial Learning
Cannabinoid agonists WIN55,212-2 and AM404 signi-
cantly impaired LTP in the Schaffer collateral-CA1 projection
FIGURE 4. CB1 receptor antagonist and agonists differen-
tially affect the induction of LTD. (a) AM404 (10 mg, n 5 5)
injected i.p. 20 min before application of low-frequency stimula-
tion (LFS) signicantly facilitated LTD in the Schaffer collat-
eral-CA1 pathway compared with all the groups (*P < 0.05,
AM404 differs from vehicle (n 5 9) and AM251; **P < 0.01,
AM404 differs from WIN). WIN55,212-2 (WIN 0.5 mg, n 5 8)
and AM251 (2 mg, n 5 7) had no effect on the induction of
LTD. Inset: representative traces in the CA1 for vehicle (upper
traces) and AM404 10 mg (lower traces) groups taken before
(black) and 90 min after (gray) LFS to the Schaffer collateral
(calibration: 0.2 mV, 10 ls). (b) Rats were i.p. injected with
vehicle, or AM251 (2 mg) or WIN55,212-2 (0.5 mg) or AM404
(10 mg) (n 5 5 each), and eld potentials were recorded in
response to a test stimulation (0.1 Hz). There was no signicant
difference between the groups, suggesting no effect of the drugs
on neural transmission.
CANNABINOIDS FACILITATE EXTINCTION AND IMPAIR LTP 1135
Hippocampus
in the doses tested here. Indeed, previous studies have shown
that exogenous cannabinoid receptor activation and endogenous
ligands of cannabinoid receptors block eld potential LTP in
the Schaffer collateral-CA1 eld in a slice preparation (Now-
icky et al., 1987; Collins et al., 1994, 1995; Terranova et al.,
1995; Stella et al., 1997).
LTP impairment occurred in the absence of any effect on
neural transmission, supporting other studies that examined the
effects of cannabinoids on plasticity (Stella et al., 1997; Mato
et al., 2004; Hoffman et al., 2007). Interestingly, AM251 also
caused a signicant impairment of LTP induction. De Oliveira
Alvares et al. (2006) have demonstrated impairment of LTP in
a CA1 slice preparation following AM251 administration.
Sokal et al. (2008) found that the CB1 receptor antagonist
SR141716A blocked the potentiation of the fEPSP slope
observed following HFS to the perforant path. However, other
studies conducted on hippocampal slices of the Schaffer collat-
eral-CA1 synapses have shown that CB1 blockade favors LTP
in the hippocampus (Slanina et al., 2005) and that mice lack-
ing CB1 receptors show enhanced LTP (Bohme et al., 2000).
However, in the study by Slanina et al. (2005), the drug was
present throughout the experiment and LTP was elicited by
moderate stimulations (20 or 50 pulses). Thus, the discrepan-
cies with our ndings could result from the examination of
eld potential in an intact rat model versus slices, or from vari-
ous methodological issues, such as different stimulation proto-
cols, different drug doses, etc.
A possible mechanism for cannabinoid receptor activation
inhibition of LTP induction in the CA1 may involve decreased
GABAergic and glutamatergic transmission in the hippocam-
pus; CB1 receptor activation inhibits glutamatergic synaptic
transmission presynaptically in cultured hippocampal neurons
(Shen et al., 1996; Misner and Sullivan, 1999; Sullivan, 1999)
and inhibits GABA release from axon terminals of CCK-con-
taining basket interneuron cells (Katona et al., 1999; Hoffman
and Lupica, 2000). Decreased glutamate release abolishes LTP,
and exogenous application of cannabinoids may prevent hippo-
campal LTP via such a mechanism (Sullivan, 2000). Cannabi-
noids also act to decrease the activity of local GABAergic inhib-
itory networks within the CA1, leading to disinhibition of glu-
tamatergic pyramidal neurons (Cobb et al., 1995; Ylinen et al.,
1995; Katona et al., 1999). Indeed, it has been suggested that
in the hippocampus, GABAergic axon terminals express strik-
ingly high levels of CB1 receptors (Katona et al., 1999; Tsou
et al., 1999; Hoffman and Lupica, 2000), when compared to
glutamatergic boutons (Katona et al., 2006). Thus, the specic
interaction between GABAergic and glutamatergic mechanisms
following exogenous CB1 receptor activation needs further
investigation before a clear conclusion can be drawn.
As for the impairing effects of the CB1 receptor antagonist
on LTP induction, this may be mediated by the effects of
AM251 over GABAergic interneurons that (indirectly) modu-
late the glutamatergic, LTP-bearing pyramidal cells (De Oli-
veira Alvares et al., 2006). In support, it has been shown that
AM251 blocks WIN55,212-2-induced inhibition of GABAer-
gic evoked inhibitory postsynaptic currents (IPSCs), but not of
glutamatergic excitatory postsynaptic currents (EPSCs). Thus,
cannabinoid receptor activation and deactivation may impair
long-term synaptic plasticity by possibly modulating neuro-
transmitter (i.e., GABA) release from presynaptic terminals.
Since the effects of the drugs on LTP do not correspond to
their behavioral effects in the IA task, we aimed at examining
their effects in a hippocampal task that is not fear-related. Can-
nabinoid agonists and antagonist impair task performance in
the water maze when administered i.p. before training,
although it should be taken into consideration that a relatively
low number of animals per group were used. In any case, our
results corroborate other studies using systemic agents in the
water maze task (Da Silva and Takahashi, 2002). Cannabinoids
agonists and antagonist were also found to impair performance
in the spatial task when microinjected locally into the CA1.
Yet, it should be noted that the impairment in performance in
the intra-CA1 experiment was more robust, compared with the
impairment in performance in the systemic experiment, in all
the drugs examined. This could be the result of differences in
drug doses between the intracranial and i.p. injections. In any
case, it should be taken into consideration that these results
apply for the specic doses tested. Taken together, the results
show that cannabinoid receptor activation and deactivation
impaired both hippocampal spatial learning and LTP.
Effect of Cannabinoids on the Induction of LTD
Our ndings indicate that AM404 signicantly facilitated
CA1 LTD, while AM251 and WIN55,212-2 had no effect.
Misner and Sullivan (1999) reported that cannabinoid receptor
activation blocks the induction of LTP and LTD of CA1 eld
potentials by reducing presynaptic neurotransmitter release to a
level below that required to depolarize the postsynaptic mem-
brane and so relieve Mg21 blockade of NMDA receptors.
Chevaleyre and Castillo (2003) have demonstrated that repeti-
tive activation of presynaptic glutamatergic bers in CA1 slices
can induce LTD of hippocampal inhibitory synapses via retro-
grade cannabinoid signaling. Their results indicate that LTD
induction requires the activation of CB1 receptors, since LTD
was blocked by inhibiting endocannabinoid production. How-
ever, it is important to note that pharmacological administra-
tion of exogenous cannabinoids may lead to different actions
than those induced by the endogenous agents of the endocan-
nabinoid system (Cota et al., 2006; Marsicano and Lutz, 2006;
Pagotto et al., 2006).
Unlike the agonist WIN55,212-2, AM404 does not directly
interact with CB1 receptors, but rather inhibits endocannabi-
noid uptake. WIN55,212-2 and AM251 act on CB1 receptors
directly, respectively, enhancing or suppressing their activity,
and so affecting GABAergic neurotransmission (Pertwee and
Ross, 2002). It is therefore possible that the different modes of
action of the different drugs, together with the extended
time period of low frequency stimulation, resulted in differen-
tial effects for exogenous cannabinoids on LTP and LTD. Yet,
these differential effects may also suggest that cannabinoids
1136 ABUSH AND AKIRAV
Hippocampus
have a specic independent role in LTD that differs from their
role in LTP.
Most of our knowledge about cannabinoids and activity-
dependent changes in synaptic strength comes from studies per-
formed at excitatory synapses, largely using acute hippocampal
slices as the experimental model (Chevaleyre et al., 2006). Our
use here of exogenous pharmacology to study the effects of can-
nabinoid activation and blockade on plasticity in an anesthe-
tized rat indicates that the experimental conditions used are
crucial to reveal a specic behavioral or cellular prole of the
responses induced by the cannabinoid system (Katona et al.,
1999). Moreover, since we used systemic infusions, the possibil-
ity that other modulatory brain areas (for example, the amyg-
dala; Akirav and Richter-Levin, 2002) may have affected the
induction of LTP and LTD should be taken into consideration.
SUMMARY
This study was designed to examine the potential effects of
the cannabinoid system on hippocampal-dependent memory
and plasticity. To our knowledge, this is the rst comprehensive
study to use the same pharmacological cannabinoid agents to
test conditioning and extinction of a fear-related learning task
compared with a non-fear-related task, as well as LTP and LTD
in the CA1. Taken together, these ndings clearly demonstrate
that the cannabinoid system has diverse effects on hippocam-
pal-dependent memory and plasticity that cannot be catego-
rized simply into an impairing or an enhancing effect on can-
nabinoid activation and deactivation, respectively.
Importantly, since it has been shown that a drug that facili-
tates extinction of conditioned fear in laboratory animals may
be utilized with success in humans (Ressler et al., 2004), our
ndings may provide preclinical support to the claim that the
cannabinoid system might represent a therapeutic target for the
treatment of diseases characterized by impaired extinction of
fear, such as PTSD and phobia. Studies examining the involve-
ment of cannabinoids in memory processes should take into
consideration whether the benets of the clinical use of canna-
binoids outweigh the risks of possible memory impairments.
REFERENCES
Akirav I, Richter-Levin G. 2002. Mechanisms of amygdala modulation
of hippocampal plasticity. J Neurosci 22:99129921.
Akirav I, Sandi C, Richter-Levin G. 2001. Differential activation of
hippocampus and amygdala following spatial learning under stress.
Eur J Neurosci 14:719725.
Berrendero F, Sepe N, Ramos JA, Di Marzo V, Fernandez-Ruiz JJ.
1999. Analysis of cannabinoid receptor binding and mRNA expres-
sion and endogenous cannabinoid contents in the developing rat
brain during late gestation and early postnatal period. Synapse
33:181191.
Bohme GA, Laville M, Ledent C, Parmentier M, Imperato A. 2000.
Enhanced long-term potentiation in mice lacking cannabinoid CB1
receptors. Neuroscience 95:57.
Chevaleyre V, Castillo PE. 2003. Heterosynaptic LTD of hippocampal
GABAergic synapses: A novel role of endocannabinoids in regulat-
ing excitability. Neuron 38:461472.
Chevaleyre V, Takahashi KA, Castillo PE. 2006. Endocannabinoid-medi-
ated synaptic plasticity in the CNS. Annu Rev Neurosci 29:3776.
Chhatwal JP, Ressler KJ. 2007. Modulation of fear and anxiety by the
endogenous cannabinoid system. CNS Spectr 12:211220.
Chhatwal JP, Davis M, Maguschak KA, Ressler KJ. 2005. Enhancing
cannabinoid neurotransmission augments the extinction of condi-
tioned fear. Neuropsychopharmacology 30:516524.
Cobb SR, Buhl EH, Halasy K, Paulsen O, Somogyi P. 1995. Synchro-
nization of neuronal activity in hippocampus by individual
GABAergic interneurons. Nature 378:7578.
Collins DR, Pertwee RG, Davies SN. 1994. The action of synthetic
cannabinoids on the induction of long-term potentiation in the rat
hippocampal slice. Eur J Pharmacol 11:R7R8.
Collins DR, Pertwee RG, Davies SN. 1995. Prevention by the canna-
binoid antagonist, SR141716A, of cannabinoid-mediated blockade
of long-term potentiation in the rat hippocampal slice. Br J Phar-
macol 115:869870.
Cota D, Tschop MH, Horvath TL, Levine AS. 2006. Cannabinoids,
opioids and eating behavior: The molecular face of hedonism?
Brain Res Rev 51:85107.
Da Silva GE, Takahashi RN. 2002. SR 141716A prevents delta 9-tet-
rahydrocannabinolinduced spatial learning decit in a Morris-type
water maze in mice. Prog Neuropsychopharmacol Biol Psychiatry
26:321325.
De Oliveira Alvares L, De Oliveira LF, Camboim C, Diehl F, Genro
BP, Lanziotti VB, Quillfeldt JA. 2005. Amnestic effect of intrahip-
pocampal AM251, a CB1-selective blocker, in the inhibitory avoid-
ance, but not in the open eld habituation task, in rats. Neurobiol
Learn Mem 83:119124.
De Oliveira Alvares L, Genro BP, Vaz Breda R, Pedroso MF, Da Costa
JC, Quillfeldt JA. 2006. AM251, a selective antagonist of the CB1
receptor, inhibits the induction of long-term potentiation and
induces retrograde amnesia in rats. Brain Res 1075:6067.
De Oliveira Alvares L, Genro BP, Diehl F, Quillfeldt JA. 2008a. Differential
role of the hippocampal endocannabinoid system in the memory con-
solidation and retrieval mechanisms. Neurobiol Learn Mem 90:19.
De Oliveira Alvares L, Pasqualini Genro B, Diehl F, Molina VA, Quillfeldt
JA. 2008b. Opposite action of hippocampal CB1 receptors in memory
reconsolidation and extinction. Neuroscience 154:16481655.
Egashira N, Mishima K, Iwasaki K, Fujiwara M. 2002. Intracerebral
microinjections of delta 9-tetrahydrocannabinol: Search for the
impairment of spatial memory in the eight-arm radial maze in rats.
Brain Res 952:239245.
Ferrari F, Ottani A, Vivoli R, Giuliani D. 1999. Learning impairment
produced in rats by the cannabinoid agonist HU 210 in a water-
maze task. Pharmacol Biochem Behav 64:555561.
Ganon-Elazar E, Akirav I. 2009. Cannabinoid receptor activation in the
basolateral amygdala blocks the effects of stress on the conditioning
and extinction of inhibitory avoidance. J Neurosci 29:1107811088.
Hampson RE, Deadwyler SA. 1999. Cannabinoids, hippocampal
function and memory. Life Sci 65:715723.
Hoffman AD, Lupica CR. 2000. Mechanisms of cannabinoid inhibi-
tion of GABAA synaptic transmission in the hippocampus. J Neu-
rosci 20:24702479.
Hoffman AF, Oz M, Yang R, Lichtman AH, Lupica CR. 2007.
Opposing actions of chronic Delta9-tetrahydrocannabinol and can-
nabinoid antagonists on hippocampal long-term potentiation.
Learn Mem 14:6374.
Iwasaki K, Matsumoto Y, Fujiwara M. 1992. Effect of nebracetam on the
disruption of spatial cognition in rats. Jpn J Pharmacol 58:117126.
Katona I, Sperlagh B, Sik A, Kafalvi A, Vizi ES, Mackie K, Freund
TF. 1999. Presynaptically located CB1 cannabinoid receptors regu-
late GABA release from axon terminals of specic hippocampal
interneurons. J Neurosci 19:45444558.
CANNABINOIDS FACILITATE EXTINCTION AND IMPAIR LTP 1137
Hippocampus
Katona I, Urban GM, Wallace M, Ledent C, Jung KM, Piomelli D,
Mackie K, Freund TF. 2006. Molecular composition of the endocan-
nabinoid system at glutamatergic synapses. J Neurosci 26:56285637.
Kim JJ, DeCola JP, Landeira-Fernandez J, Fanselow MS. 1991. N-
methyl-D-aspartate receptor antagonist APV blocks acquisition but
not expression of fear conditioning. Behav Neurosci 105:126133.
Kobilo T, Hazvi S, Dudai Y. 2007. Role of cortical cannabinoid CB1
receptor in conditioned taste aversion memory. Eur J Neurosci
25:34173421.
Laviolette SR, Grace AA. 2006a. Cannabinoids potentiate emotional
learning plasticity in neurons of the medial prefrontal cortex
through basolateral amygdala inputs. J Neurosci 26:64586468.
Laviolette SR, Grace AA. 2006b. The roles of cannabinoid and dopamine
receptor systems in neural emotional learning circuits: Implications for
schizophrenia and addiction. Cell Mol Life Sci 63:15971613.
Lichtman AH. 2000. SR 141716A enhances spatial memory as
assessed in a radial-arm maze task in rats. Eur J Pharmacol
404:175179.
Lichtman AH, Dimen KR, Martin BR. 1995. Systemic or intrahippo-
campal cannabinoid administration impairs spatial memory in rats.
Psychopharmacology 119:282290.
Lutz B. 2007. The endocannabinoid system and extinction learning.
Mol Neurobiol 36:92101.
Marsicano G, Lutz B. 2006. Neuromodulatory functions of the endo-
cannabinoid system. J Endocrinol Invest 29:2746.
Marsicano G, Wotjak CT, Azad SC, Bisogno T, Rammes G, Cascio
MG, Hermann H, Tang J, Hofmann C, Zieglgansberger W, Di
Marzo V, Lutz B. 2002. The endogenous cannabinoid system con-
trols extinction of aversive memories. Nature 1:530534.
Martin WJ, Cofn PO, Attias E, Balinsky M, Tsou K, Walker JM.
1999. Anatomical basis for cannabinoid-induced antinociception as
revealed by intracerebral microinjections. Brain Res 822:237242.
Mato S, Chevaleyre V, Robbe D, Pazos A, Castillo PE, Manzoni OJ.
2004. A single in vivo exposure to delta 9THC blocks endocanna-
binoid-mediated synaptic plasticity. Nat Neurosci 7:585586.
Milad MR, Quirk GJ. 2002. Neurons in medial prefrontal cortex sig-
nal memory for fear extinction. Nature 420:7074.
Misner DL, Sullivan JM. 1999. Mechanism of cannabinoid effects on
long-term potentiation and depression in hippocampal CA1 neu-
rons. J Neurosci 19:67956805.
Moreira FA, Aguiar DC, Guimaraes FS. 2007. Anxiolytic-like effect of
cannabinoids injected into the rat dorsolateral periaqueductal gray.
Neuropharmacology 52:958965.
Nowicky AV, Teyler TJ, Vardaris RM. 1987. The modulation of long-
term potentiation by delta-9-tetrahydrocannabinol in the rat hippo-
campus, in vitro. Brain Res Bull 19:663672.
Pagotto U, Marsicano G, Cota D, Lutz B, Pasquali R. 2006. The
emerging role of the endocannabinoid system in endocrine regula-
tion and energy balance. Endocr Rev 27:73100.
Pamplona FA, Takahashi RN. 2006. WIN 552122 impairs contextual
fear conditioning through the activation of CB1 cannabinoid
receptors. Neurosci Lett 397:8892.
Pamplona FA, Prediger RD, Pandolfo P, Takahashi RN. 2006. The
cannabinoid receptor agonist WIN55,2122 facilitates the extinc-
tion of contextual fear memory and spatial memory in rats. Psy-
chopharmacology 188:641649.
Pamplona FA, Bitencourt RM, Takahashi RN. 2008. Short- and long-
term effects of cannabinoids on the extinction of contextual fear
memory in rats. Neurobiol Learn Mem 90:290293.
Paxinos G, Watson C. 1998. A Stereotaxic Atlas of the Rat Brain.
New York: Academic Press.
Pertwee RG, Ross RA. 2002. Cannabinoid receptors and their ligands.
Prostaglandins Leukot Essent Fatty Acids 66:101121.
Ressler KJ, Rothbaum BO, Tannenbaum L, Anderson P, Graap K, Zimand
E, Hodges L, Davis M. 2004. Cognitive enhancers as adjuncts to psy-
chotherapy: Use of D-cycloserine in phobic individuals to facilitate
extinction of fear. Arch Gen Psychiatry 61:11361144.
Riedel G, Davies SN. 2005. Cannabinoid function in learning, mem-
ory and plasticity. Handb Exp Pharmacol 168:445477.
Roozendaal B, Hahn EL, Nathan SV, de Quervain DJ, McGaugh JL.
2004. Glucocorticoid effects on memory retrieval require concur-
rent noradrenergic activity in the hippocampus and basolateral
amygdala. J Neurosci 24:81618169.
Shen M, Piser TM, Seybold VS, Thayer SA. 1996. Cannabinoid re-
ceptor agonists inhibit glutamatergic synaptic transmission in rat
hippocampal cultures. J Neurosci 16:43224334.
Sink KS, Vemuri VK, Olszewska T, Makriyannis A, Salamone JD.
2008. Cannabinoid CB1 antagonists and dopamine antagonists
produce different effects on a task involving response allocation
and effort-related choice in food-seeking behavior. Psychopharma-
cology (Berl) 196:565574.
Slanina KA, Roberto M, Schweitzer P. 2005. Endocannabinoids
restrict hippocampal long term potentiation via CB1. Neurophar-
macology 49:660668.
Sokal DM, Benetti C, Girlanda E, Large CH. 2008. The CB1 recep-
tor antagonist, SR141716A, prevents high-frequency stimulation-
induced reduction of feedback inhibition in the rat dentate gyrus
following perforant path stimulation in vivo. Brain Res 1223:50
58.
Stella N, Schweitzer P, Piomelli D. 1997. A second endogenous canna-
binoid that modulates long-term potentiation. Nature 388:773
778.
Suenaga T, Ichitani Y. 2004. Effects of intrahippocampal
WIN55,2122 on T-maze delayed alternation in rats. Jpn J Animal
Psychol 54:143.
Sullivan JM. 1999. Mechanisms of cannabinoid-receptor-mediated in-
hibition of synaptic transmission in cultured hippocampal pyrami-
dal neurons. J Neurophysiol 82:12861294.
Sullivan JM. 2000. Cellular and molecular mechanisms underlying
learning and memory impairments produced by cannabinoids.
Learn Mem 7:132139.
Suzuki A, Josselyn SA, Frankland PW, Masushige S, Silva AJ, Kida S.
2004. Memory reconsolidation and extinction have distinct tempo-
ral and biochemical signatures. J Neurosci 24:47874795.
Terranova JP, Michaud JC, Le Fur G, Soubrie P. 1995. Inhibition of
long-term potentiation in rat hippocampal slices by anandamide
and WIN552122: Reversal by SR141716 A, a selective antagonist
of CB1 cannabinoid receptors. Naunyn Schmiedebergs Arch Phar-
macol 352:576579.
Terranova JP, Storme JJ, Lafon N, Per io A, Rinaldi-Carmona M, Le
Fur G Soubrie P. 1996. Improvement of memory in rodents by the
selective CB1 cannabinoid receptor antagonist, SR 141716. Psycho-
pharmacology 126:165172.
Tinsley MR, Quinn JJ, Fanselow MS. 2004. The role of muscarinic
and nicotinic cholinergic neurotransmission in aversive condition-
ing: Comparing pavlovian fear conditioning and inhibitory avoid-
ance. Learn Mem 11:3542.
Tsou K, Mackie K, Sanudo-Pena MC, Walker JM. 1999. Cannabinoid
CB1 receptors are localized primarily on cholecystokinin- contain-
ing GABAergic interneurons in the rat hippocampal formation.
Neuroscience 93:969975.
Varvel SA, Hamm RJ, Martin BR, Lichtman AH. 2001. Differential
effects of delta9-THC on spatial reference and working memory in
mice. Psychopharmacology 157:142150.
Varvel SA, Wise LE, Niyuhire F, Cravatt BF, Lichtman AH. 2007. In-
hibition of fatty-acid amide hydrolase accelerates acquisition and
extinction rates in a spatial memory task. Neuropsychopharmacol-
ogy 32:10321041.
Viveros MP, Marco EM, Llorente R, Lopez-Gallardo M. 2007. Endo-
cannabinoid system and synaptic plasticity: Implications for emo-
tional responses. Neural Plast 2007:52908.
Ylinen A, Soltesz I, Bragin A, Penttonen M, Sik A, Buzsaki G. 1995. Intra-
cellular correlates of hippocampal theta rhythm in identied pyramidal
cells, granule cells, and basket cells. Hippocampus 5:7890.
1138 ABUSH AND AKIRAV
Hippocampus