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Abstract We have previously reported that a single
injection of an ultra-low dose of delta-9-tetrahydrocanna-
binol (THC; the psychoactive ingredient of marijuana)
protected the brain from pentylenentetrazole (PTZ)-
induced cognitive deficits when applied 1–7 days before or
1–3 days after the insult. In the present study we expanded
the protective profile of THC by showing that it protected
mice from cognitive deficits that were induced by a variety
of other neuronal insults, including pentobarbital-induced
deep anesthesia, repeated treatment with 3,4 methylene-
dioxymethamphetamine (MDMA; ‘‘ecstasy’’) and expo-
sure to carbon monoxide. The protective effect of THC
lasted for at least 7 weeks. The same ultra-low dose of
THC (0.002 mg/kg, a dose that is 3–4 orders of magnitude
lower than the doses that produce the known acute effects
of the drug in mice) induced long-lasting (7 weeks) mod-
ifications of extracellular signal–regulated kinase (ERK)
activity in the hippocampus, frontal cortex and cerebellum
of the mice. The alterations in ERK activity paralleled
changes in its activating enzyme MEK and its inactivating
enzyme MKP-1. Furthermore, a single treatment with the low dose of THC elevated the level of pCREB (phos-
phorylated cAMP response element–binding protein) in the
hippocampus and the level of BDNF (brain-derived neu-
rotrophic factor) in the frontal cortex. These long-lasting
effects indicate that a single treatment with an ultra-low
dose of THC can modify brain plasticity and induce long-
term behavioral and developmental effects in the brain.
Keywords Cannabinoid  Neuroprotection 
Preconditioning  Cognitive deficit 
Extracellular signal–regulated kinase (ERK)
Cannabis, though considered a mild, ‘‘soft’’ drug without
long-lasting negative effects, has been shown to cause
long-term cognitive deficits in chronic users manifested as
impairment in attention, memory or executive functions
(Solowij et al. 1995; Block 1996; Ehrenreich et al. 1999;
McHale and Hunt 2008; Abdullaev et al. 2010; Battisti
et al. 2010; Lundqvist 2010; Coullaut-Valera et al. 2011).
A meta-analysis that examined the non-acute effects of
cannabis concluded that there might be decrements in the
ability to learn and remember new information in chronic
users (Grant et al. 2003). Other studies showed emotional
effects such as anxiety, depression (Reilly et al. 1998;
Troisi et al. 1998; Bovasso 2001; Patton et al. 2002; Gruber
et al. 2009) and lack of motivation (Kouri et al. 1995).
fMRI studies indicated that even when cognitive deficits
are not observed, chronic users compensate by mobilizing
more neuronal resources in order to perform the required
cognitive function (Kanayama et al. 2004; Jager et al.
2006). Furthermore, MRI studies showed a reduction in
white and gray matter in the cerebellum of chronic cannabis users (Cohen et al. 2012; Solowij et al. 2011).
Studies with human subjects may inevitably be hampered
by various confounding factors such as drug residues,
abstinence effects or methodological drawbacks (Solowij
et al. 2002; Grant et al. 2003; Fried et al. 2005), while
animal studies enable better controlled studies. Indeed,
consistent findings of long-lasting cognitive deficits were
demonstrated following chronic exposure of laboratory
animals to cannabinoid drugs. Chronic exposure of rats to
D9 -tetrahydrocannabinol (THC) resulted in persistent
reduction in maze learning (Fehr et al. 1976; Stiglick and
Kalant 1982a, 1983) and in differential reinforcement
responding (Stiglick and Kalant 1982b). Repeated expo-
sure of rats to cannabinoid agonists during perinatal, ado-
lescent or early adult ages produced long-lasting deficits in
working memory and social interaction (O’Shea et al.
2006) as well as in spatial learning (Rubino et al. 2009) and
caused anxiety and depression in rats (Rubino et al. 2008;
Bambico et al. 2010). Morphological changes in the hip-
pocampus of rats that were chronically treated with can-
nabinoids, including neuronal death and reduced synaptic
density and dendritic length of pyramidal neurons, were
also reported (Scallet et al. 1987; Landfield et al. 1988;
Scallet 1991; Lawston et al. 2000; Rubino et al. 2009).
In contrast to the above reports, other studies have
suggested that cannabinoids may have neuroprotective
properties [for reviews see (Guzman et al. 2002;
Mechoulam et al. 2002; Sarne and Mechoulam 2005; van
der Stelt and Di Marzo 2005)]. Acute administration of
cannabinoid agonists was found to protect against global
and focal ischemic damage (Nagayama et al. 1999), against
ouabain-induced excitotoxicity (van der Stelt et al. 2001a,
b), against severe closed head injury (Panikashvili et al.
2001; Mauler et al. 2003) and against MDMA (3,4
methylenedioxymethamphetamine) neurotoxicity (Tourino
et al. 2010). Other studies suggested neuroprotective
effects of cannabinoids in neurodegenerative pathologies
such as multiple sclerosis, Alzheimer’s, Huntington’s and
Parkinson’s diseases (Gowran et al. 2011; Sagredo et al.
2011). It was even suggested that the endogenous can-
nabinoid system has a physiological role in neuroprotection
(Guzman et al. 2001; Mechoulam et al. 2002; Marsicano
et al. 2003; Karanian et al. 2007).
The neuroprotective properties of cannabinoids are
attributed, among other factors, to their ability to suppress
voltage-dependent calcium channels (Mackie and Hille
1992) and consequently to attenuate the release of gluta-
mate (Shen et al. 1996). In vitro findings, however, have
shown that very low concentrations of cannabinoids can
potentiate, rather than suppress, calcium entry into cells
(Okada et al. 1992; Rubovitch et al. 2002), thus suggesting
that very low doses of cannabinoid drugs may be neurotoxic in vivo. Indeed, in recent studies we have
shown that a single extremely low dose of THC (0.002 mg/
kg, a dose that is 3–4 orders of magnitude lower than the
doses that produce the known acute effects of the drug in
mice) significantly deteriorated the cognitive performance
of mice that were tested 3 weeks and up to 5 months
following the injection, in various behavioral assays
that assess different aspects of learning and memory
(Tselnicker et al. 2007; Senn et al. 2008; Amal et al. 2010).
These findings led to the idea that this ultra-low dose of
THC, which induces minor damage to the brain, may
activate preconditioning and/or postconditioning mecha-
nisms and thus will protect the brain from more severe
insults (Sarne et al. 2011). Indeed, our recent findings
support this assumption and show that treatment with
extremely low doses of THC, several days before or after
PTZ (pentylenentetrazole)-induced seizures, provides
effective long-term cognitive neuroprotection (Assaf et al.
The long-lasting cognitive effects of the ultra-low dose
of THC were found by us to be accompanied by a delayed
(24 h) activation of ERK (extracellular signal–regulated
kinase) in the cerebellum (Senn et al. 2008; Amal et al.
2010). ERK has been shown to have an important role in
regulating many processes of cellular homeostasis,
including both cell survival and death (Agell et al. 2002;
Liou et al. 2003), and is considered a crucial component in
the formation of long-term memory and for the strength-
ening of synaptic connectivity in a variety of behavioral
processes (Fasano and Brambilla 2011). Several studies
claim that ERK is involved in the beneficial effects of
preconditioning in the ischemic brain (Choi et al. 2006; Jin
et al. 2006), yet other studies claim that ERK promotes
inflammation and oxidative stress (Noshita et al. 2002;
Maddahi and Edvinsson 2011). A dual effect of ERK was
also seen in stroke (Sawe et al. 2008). The dual activity of
ERK makes it a suitable neuronal marker to investigate the
long-lasting endogenous neuronal mechanism(s) that is
modulated by the low dose of THC that induced both
cognitive deficits (Tselnicker et al. 2007; Senn et al. 2008;
Amal et al. 2010) and neuroprotection (Assaf et al. 2011).
In the present study we followed the long-term
(7 weeks) effects of an ultra-low dose of THC on the entire
ERK signaling system, including its upstream and down-
stream effectors, in different brain regions. We also sear-
ched for biochemical interactions between the insult-
inducing agent (PTZ) and the protecting factor (THC). In
order to explore how general the protective effect of THC
is, we tested its ability to protect the mice from various
different insults, including pentobarbital-induced deep
anesthesia, repeated administration of MDMA (’’ecstasy’’)
and exposure to carbon monoxide.

Experimental procedures
The study was performed on male ICR mice, 8 weeks old,
weighing 30–40 gr. The animals were housed 8–10 per
cage in the Animal Care Facility at a temperature of 21 °C
and a 14/10-h light/dark cycle, with free access to food and
water. All behavioral experiments were performed during
the light phase. The experimental protocols were approved
by the Institutional Animal Care and Use Committee of
Tel-Aviv University.
In order to reduce stress at the day of treatment, the mice
were habituated by daily injections of saline, at least 2 times
prior to the treatment. All drugs were injected intraperito-
neally (i.p) in a volume of 0.1 ml/10 g body weight. D9-
tetrahydrocannabinol (THC) (donated by NIDA, USA, and
by Prof. Mechoulam, the Hebrew University, Jerusalem)
was dissolved from a stock solution made in ethanol into a
vehicle solution consisted of 1:1:18 ethanol/cremophor
(Sigma–Aldrich)/saline and administered in a dose of
0.002 mg/kg. Pentylenentetrazole (PTZ) (Sigma-Aldrich)
was dissolved in saline and administered in a dose of 60 mg/
kg. Pentobarbital (pento) (Pental veterinary, CTS, Chemical
industries) was dissolved in saline and administered in a
dose of 100 mg/kg. This dose of pentobarbital caused no
mortality, and the mice were asleep for about 8 h after the
injection. MDMA (3,4 methylenedioxymethamphetamine)
was dissolved in saline and injected daily throughout three
consecutive days in a dose of 10 mg/kg. Mice were exposed
to carbon monoxide (CO) in a tightly closed glass chamber
for 12 s thrice with 45-min intervals between exposures.
Behavioral tests
Double-blind behavioral tests have been carried out
3–7 weeks after the experimental treatment (pento, CO or
MDMA with or without THC). Each experiment consisted
of 4 groups of treated mice and their matched controls. In
order to carry out behavioral tests of treated and control
mice on the same day and under the same conditions,
experiments were repeated several times with small groups
of mice, and results were combined for statistical analysis.
Each group of mice underwent the three behavioral tests
(see below), and representative experiments are shown.
Open field
Motor activity was tested in an open field that consisted of
a black plastic arena (60 cm 9 60 cm) whose floor was divided into 10 cm 9 10 cm squares. Each mouse was
introduced to the field, and the number of squares that it
crossed during a 5-min session was recorded. The arena
was cleaned with alcohol after each mouse.
The dry maze
The dry maze (‘‘oasis test’’) is a land-based spatial learning
assay that was designed to approximate the spatial learning
demands required by the Morris water maze. The test was
performed according to Clark et al. (2005), with modifi-
cations (Amal et al. 2010). The maze consisted of a white
plastic arena (200 cm in diameter) with 20 wells arranged
in 3 concentric circles. Each well contained 0.27 ml of
water. The maze was situated in a room that contained
various visual clues. The mice were allowed to drink freely
only 1 h per day starting from 3 days before the experi-
ment and all along the experiment. Each experiment
comprised two parts. In the first part (‘‘training stage’’) all
the wells were filled with water. The mouse was introduced
into the arena at different starting points, with its head
turned toward the wall of the arena, and the time the mouse
took to find a well and drink from it was recorded. The
‘‘training stage’’ consisted of 3 trials of 3 min each. On the
second part of the experiment (the ‘‘test stage’’, trials
4–10), the mouse was introduced into the same arena, with
only one of the 20 wells filled with water (the same single
well in all the trials). Each mouse was introduced into the
arena twice daily, and the arena was cleaned with alcohol
after each trial. The time the mouse took to locate the
single well that was filled with water and to drink from it
was recorded. If the mouse failed to locate the well within
3 min, it was gently guided to the filled well.
Object recognition test
The test utilizes the tendency of mice to explore novel
objects. The assay consisted of two parts: a familiarization
session and a test session. Twenty-four hour before the test,
the mice were allowed to explore the black plastic arena
without objects for 5 min. During the first session, the mice
were left to explore for 5 min two identical objects that
were located 10 cm from the side walls. Twenty-four hour
later, the mice were introduced to the arena for the test
session in which one of the familiar objects was replaced
by a novel object. Exploration was defined as directing the
nose of the mouse to the object at a distance of B1 cm and/
or touching the object with the nose. The time spent by the
mouse in exploring each object was recorded for 5 min,
and relative time of exploration was calculated as the time
spent exploring the object over total exploration time for
both objects. The arena and the objects were cleaned with
alcohol after each trial.

Biochemical procedures
Tissue preparation
Mice were killed by cervical dislocation after completing
the behavioral tests, 7 weeks after treatment. The brain was
removed and the cerebellum, hippocampus, frontal cortex
and occipital cortex were dissected out and immediately
frozen in liquid nitrogen and stored at -80 °C. Each
sample was homogenized in 200–400 ll of ice-cold
extraction buffer containing 10 mM potassium phosphate,
pH 7.5, 10 mM MgCl2, 5 mM EDTA, 1 mM EGTA,
1 mM sodium orthovanadate, 2 mM DTT, 1 % Triton
X-100, 50 mM b-glycerophosphate, 0.5 % protease inhib-
itor cocktail (Sigma–Aldrich) and 1 % phosphatase inhib-
itor cocktail 1 (Sigma–Aldrich). The samples were
homogenized by hand in small glass homogenizers, and the
homogenates were centrifuged at 15,0009g at 4 °C for
10 min. The supernatants were stored at -80 °C. Protein
concentration was determined with the Bradford Reagent
Western blotting
Homogenate aliquots containing 60 lg of total protein
made in sample buffer [50 mM Tris (pH 6.8), 10 % glyc-
erol, 5 % mercaptoethanol, 2 % SDS and bromophenol
blue (Sigma-Aldrich)] were loaded and separated on 10 %
SDS–polyacrylamide gels and then transferred to nitro-
cellulose membranes (Whatman, Schleicher & Schuell,
Dassel, Germany). The membranes were blocked in
10 mM Tris–HCl (pH 7.4), 135 mM NaCl and 0.1 %
Tween-20 (TTBS) containing 5 % fat-free milk powder for
1 h at room temperature and then incubated overnight at
4 °C with either a mouse monoclonal antibody against
phosho(Tyr 204)-ERK1/2 (1:600; Santa Cruz Biotechnol-
ogy Inc., Santa Cruz, CA, USA) or a rabbit monoclonal
antibody against p-CREB (1:500; Santa Cruz Biotechnol-
ogy Inc., Santa Cruz, CA, USA) or a rabbit monoclonal
antibody against BDNF (1:400; Santa Cruz Biotechnology
Inc., Santa Cruz, CA, USA). The membranes were then
washed and incubated for 60 min at room temperature with
horseradish peroxidase-labeled goat anti-mouse antibody
(1:10,000; Santa Cruz Biotechnology Inc.) or goat anti-
rabbit (1:10,000; Santa Cruz Biotechnology Inc.) and
developed using an enhanced chemiluminescence (ECL)
reagent. To determine total ERK1/2 levels, the membranes
were stripped by 10-min incubation in 0.1 M NaOH con-
taining 0.2 % SDS and reprobed for 60 min in room tem-
perature with a rabbit polyclonal antibody raised against
ERK (1:1,000; Santa Cruz Biotechnology Inc.) and then
washed and incubated for 60 min at room temperature with
horseradish peroxidase-labeled goat anti-rabbit antibody (1:10,000; Santa Cruz Biotechnology Inc.). The films were
scanned, and the optical density of the relevant band was
analyzed using TINA2.07 software. To allow comparison
between different films, the optical density of the bands
was expressed relative to the average of control samples in
each film.
Data analysis
All results are presented as mean ± SEM. Comparison of
two groups was carried out by Student’s t test and multiple
comparisons by analysis of variance (ANOVA) followed
by Tukey’s post hoc test. Level of significance was set at
p B 0.05.
An ultra-low dose of THC protects against different
insults that cause cognitive deficits
In our previous study (Assaf et al. 2011), we have shown
that an ultra-low dose of THC applied 1–7 days before or
1–3 days after pentylenentetrazole (PTZ) protected against
cognitive deficits that were induced by PTZ. In order to test
how common the protective effect of THC was, we intro-
duced the mice, in the present study, to three different other
insults that caused cognitive deficits. First we tested whe-
ther THC, applied 24 h before pentobarbital (pento,
100 mg/kg in saline), protected against the cognitive
damage of the hypnotic drug. The experiment consisted of
4 groups of mice (control, THC, pento and THC ? pento)
with 8–10 mice per group. The mice were tested in the
object recognition assay 3 weeks after the treatment
(Fig. 1). In the test session of the object recognition assay,
control mice significantly preferred the new object, as
expected (t test, p \ 0.05). THC-treated mice did not show
behavioral deficits and similarly preferred the new object
(t test, p \ 0.05). Mice treated with pento failed to disso-
ciate between the two objects and did not prefer the new
object. However, mice that had been treated with THC
24 h before pento preferred significantly the new object
(t test, p \ 0.05) and behaved similar to the control group.
Next we exposed mice to 100 % carbon monoxide (CO)
and evaluated their behavior 3 weeks later. The experiment
consisted of 4 groups of mice: control, mice exposed to
CO, mice injected with THC and mice exposed to CO 48 h
after THC (14–21 mice per group). The mice were evalu-
ated 3 weeks later by the dry maze test. As depicted in
Fig. 2a, in the ‘‘training stage’’ (trials 1–3), when all the
wells were filled with water, the thirsty mice rapidly
learned to drink out of the wells and there was no differ-
ence between the performance of the four groups. In the second part (the ‘‘test stage’’; trials 4–10), when only one
well was filled with water and the mice had to learn and
remember its location, the four groups diverged and a two-
way ANOVA indicated a significant difference between the
groups [F(3,489) = 8.974, p \ 0.05]. It took a signifi-
cantly longer time for the CO group to find the single full
well than for the control group (Tukey’s post hoc test,
p \ 0.05). The THC group also spent more time finding the
full well than the control group, but this difference was not
statistically significant. Mice that had been injected with
THC 48 h before the exposure to CO behaved almost as the
control group and found the full well significantly faster
than the mice treated with CO (Tukey p \ 0.05). Figure 2b
presents the mean latency of the four groups at steady state
(trials 8–10). One-way ANOVA revealed a significant
effect of treatment [F(3,209) = 8.621, p \ 0.05] with a
significant difference between CO and control, THC and
control, and CO and THC ? CO groups (Tukey p \ 0.05),
but no difference was found between THC ? CO and
control mice. The results indicate that treating the mice
with an ultra-low dose (0.002 mg/kg) of THC 48 h before
their exposure to CO protected the mice from the cognitive
deficit that was induced by CO.

In the next experiment we injected the mice with 3,4
methylenedioxymeth-amphetamine (MDMA; 10 mg/kg in
saline) once a day for 3 consecutive days and tested whe-
ther THC can protect against the cognitive damage that was
caused by MDMA. In preliminary experiments we found no protection when THC was injected either 24 h before or
24 h after MDMA (data not shown) and so decided to
inject the mice with THC both 24 h before and 24 h after
MDMA. The experiment consisted of 4 groups of mice
(control, THC, MDMA and THC ? MDMA ? THC; 17
mice per group) that were examined for cognitive deficits
3 weeks following the last exposure to MDMA. Figure 3
depicts the second session of the object recognition test,
when one of the objects in the arena was replaced by a new
object. Control mice significantly preferred the new object,
as expected (t test, p \ 0.05). In contrast, neither MDMA-treated nor THC-treated mice showed a significant prefer-
ence for the new object. However, mice that were treated
with THC before and after MDMA preferred the new
object significantly (t test, p \ 0.05), similarly to the
control group (see ‘‘Discussion’’).
In all the above-described experiments, the various
insults (pento, CO and MDMA) as well as THC failed to
modify motor activity of the treated mice, as was measured
by the open field test, suggesting no motor deficits that
could interfere with the cognitive tests.
An ultra-low dose of THC induces long-term
modifications in the ERK/MAPK signaling system
Our next goal was to study the long-term biochemical
effects of a single injection of THC in various brain areas.
We have previously shown that the ultra-low dose of THC
caused an increase in ERK2 activity (phosphorylation of
ERK) in the cerebellum, 24 h after the injection (Senn
et al. 2008), followed by its decline 7 weeks later (Assaf
et al. 2011). In the current study we tested whether there
are any long-term (7 weeks) changes in ERK2 activity in
other brain regions that are known to be related to memory
and learning. In the frontal cortex and the hippocampus,
ERK2 phosphorylation rose significantly by 22 % and by
19 %, respectively, in THC-treated mice compared to their matched controls (n = 40 per group, t test, p \ 0.05)
(Fig. 4). In the same mice, ERK2 phosphorylation in the
cerebellum declined significantly by 27 % (t test,
p \ 0.05), and there was no alteration in ERK2 phos-
phorylation in the occipital cortex (Fig. 4). It is worthwhile
noting that there were no changes in total ERK in any of
these brain regions, indicating the augmentation or sup-
pression of ERK activity without affecting its synthesis or
We then tested whether the decline in ERK activity in
the cerebellum is due to the decline in the activity of its
upstream kinase MEK or due to an elevation in its specific
phosphatase MKP-1. We found that 7 weeks after a single
injection of the ultra-low dose of THC, there was a non-
significant decrease in MEK phosphorylation by 21 % as
well as a significant decrease in MKP-1 by 36 % (n = 8
per group, t test, p \ 0.05) (Fig. 5a), indicating the sup-
pression of the entire ERK/MAPK system. We also found
that ERK2 activation in the hippocampus was accompanied
by a nonsignificant elevation in phosphorylated MEK by
38 % and a significant elevation of MKP-1 by 15 % (n = 8
per group, t test, p \ 0.05), indicating the activation of the
entire ERK/MAPK system in this brain region (Fig. 5b).

We next looked for changes in proteins downstream to
ERK that are known to be related to the formation of long-
term memory and to neuroprotection. We found a signifi-
cant elevation of pCREB (phosphorylated cAMP response
element–binding protein) by 20 % in the hippocampus
(n = 32 per group, t test, p \ 0.05) but no change in its
level in the frontal cortex. On the other hand, there was a
significant elevation of BDNF (brain-derived neurotrophic
factor) by 34 % in the frontal cortex (n = 40 per group,
t test, p \ 0.05) but no change in its level in the hippo-
campus. Figure 6 depicts the elevation of pCREB and of
BDNF in the hippocampus and the frontal cortex, respec-
tively, 7 weeks after a single injection of 0.002 mg/kg of

Biochemical interaction between THC and PTZ
The behavioral interaction between THC and PTZ, in
which THC protected the mice from the cognitive damage that was induced by PTZ (Assaf et al. 2011), led us to look
for possible biochemical interactions between these two
drugs. The experiment consisted of 4 groups of mice, 16
mice per group: control, THC (0.002 mg/kg), PTZ (60 mg/
kg) and THC applied 24 h before PTZ. The mice under-
went the behavioral tests on weeks 4–6 and then were
killed 7 weeks after the treatment. In the hippocampus,
THC caused a significant elevation of 35 % in phosphor-
ylated ERK2 compared to the control group (t test,
p \ 0.05) (Fig. 7). PTZ caused a nonsignificant elevation
of 14 % in phosphorylated ERK2. When THC was applied
24 h before PTZ, the level of phosphorylated ERK2 was
significantly lower than the level in the PTZ group by 27 %
(t test, p \ 0.05), than the level in the THC group by 39 %
(t test, p \ 0.05) and than the level in the control group by
17 % (Fig. 7). We then calculated the expected additive
effect of the two drugs on ERK activity that summed up to
an elevation of 50 % compared to control (see last column
in Fig. 7). There was a clear and significant difference
between the observed and the expected effect of THC and
PTZ when applied together (see ‘‘Discussion’’).
Preconditioning is a phenomenon where a minor noxious
stimulus protects from a subsequent insult. In our previous study we showed that an ultra-low dose of D9 -tetrahydro-
cannabinol (THC) that by itself induced a minor cognitive
deficit protected against a more severe cognitive damage
caused by the epileptogenic agent pentylenentetrazole
(PTZ) (Assaf et al. 2011). In order to test how general the
neuroprotective effect of THC was, we employed, in the
present study, various insults that act through different
mechanisms and investigated the protective effect of THC.
We found that the ultra-low dose of THC protected the
mice from cognitive deficits that were caused by pento-
barbital-induced deep anesthesia, by carbon monoxide
(CO)-induced hypoxia and by the aminergic modifying
drug 3,4 methylenedioxymethamphetamine (MDMA;
‘‘ecstasy’’). As depicted in Fig. 1, when THC was applied
24 h before the anesthetic barbiturate pentobarbital, mice
were protected from the cognitive damage created by
pentobarbital itself. Though THC did not induce any
detectable cognitive damage in this specific experiment,
the protection was still evident, implying that a consider-
able damage by itself was not necessary for inducing the
compensatory mechanism(s) that led to neuroprotection.
When THC had been applied 48 h before CO (Fig. 2), a
toxic gas that binds to hemoglobin and prevents the
effective distribution of oxygen to the brain, the cognitive
damage caused by CO was abolished. In this experiment,
THC by itself also caused a mild cognitive deficit. Sur-
prisingly, mice that were treated with both insults (THC before CO) behaved not only better than CO-treated mice
but even better than the THC-treated mice. It appears as if
each insult activated compensatory mechanisms that pro-
tected against the preceding or the following insult. In this
regard it is worth mentioning that pre- and postcondition-
ing share common signaling pathways (Hausenloy and
Yellon 2009) and that each insult can protect against a
completely different type of another insult (‘‘cross-condi-
tioning’’) (Wada et al. 1999; Lin et al. 2009a, b). An
alternative explanation assumes that a single minor insult
just primes the endogenous compensating system, while
the second insult activates it. Thus, two consecutive insults
are required in order to fully activate the endogenous
protective mechanism(s). A similar situation, where an
amnesic drug (morphine) protected from the amnesic effect
of another drug (lipopolysaccharide), was recently descri-
bed (Rostami et al. 2012).
When THC was applied 24 h before and after MDMA, a
drug that potentiates the activity of serotonin, norepi-
nephrine and dopamine, the cognitive damage was abol-
ished and the mice behaved as the controls (Fig. 3). Similar
to the CO experiment, the mice that received both THC and
MDMA behaved better than single drug-treated mice,
implying a cross-protection between THC and MDMA as
well. In this experiment it was required to treat the mice
with THC both before and after MDMA in order to achieve
an effective protection. Indeed, an additive effect of pre-
and postconditioning treatments was previously demon-
strated for both neuroprotection (McMurtrey and Zuo
2010) and cardioprotection (Sato et al. 2007).
Our behavioral experiments demonstrate that precondi-
tioning with an ultra-low dose of THC is not specific for a
certain type of damage, but exerts a more general neuro-
protective effect. Cannabinoids have been previously found
by others to protect the brain from different insults. It
should be emphasized that all previous studies employed
much higher doses of the cannabinoid agent (1–10 mg/kg)
that were administered either immediately before (up to
60 min) (Nagayama et al. 1999; van der Stelt et al. 2001a,
b; Hayakawa et al. 2007; Tourino et al. 2010) or immedi-
ately after (up to 210 min) (Shouman et al. 2006;
Hayakawa et al. 2007) the insult. When the injection of the
cannabinoid was delayed by 1 h (Panikashvili et al. 2001)
or by 8 h (Shouman et al. 2006), it failed to protect the
brain. These studies are in a sharp contrast to our current
and previous (Assaf et al. 2011) results, where a long-term
protection by an ultra-low dose of THC was observed when
the drug was applied 1–7 days before or 1–3 days after the
insult. The conventional neuroprotective properties of
cannabinoids are attributed, among other factors, to their
ability to suppress voltage-gated calcium channels (Mackie
and Hille 1992) and consequently to attenuate the release
of glutamate (Shen et al. 1996). The modulation by cannabinoids of other mechanisms, such as the inhibition
of NO synthesis (Hillard et al. 1999) and the inhibition of
the release of the pro-inflammatory cytokine tumor
necrosis factor alpha (TNFa) (Facchinetti et al. 2003) was
also suggested. Other cannabinoid actions that may con-
tribute to their direct neuroprotective effects include the
induction of hypothermia (Leker et al. 2003) and vasodi-
latation (Wagner et al. 2001), and their anti-inflammatory
effects (Maresz et al. 2007; Zhang et al. 2007; Fernandez-
Ruiz et al. 2008). All these studies pointed to the acute
protective features of cannabinoids that were dependent on
the presence of the drug in a high enough concentration
close to the time of insult. We believe that the long-term
(24–48 h) neuroprotective effect of the ultra-low dose of
THC that was demonstrated in our current study was
dependent on the activation of long-lasting compensatory
mechanisms, as had been previously suggested for other
preconditioning treatments (Hausenloy and Yellon 2009).
It is not clear yet what was the immediate target of THC
that mediated its long-term protective effect. Nevertheless,
in our previous study (Senn et al. 2008) we found that
SR141716A completely blocked the long-lasting deterio-
rating effect of the ultra-low dose THC, suggesting the
involvement of CB1 cannabinoid receptors.
The long-term protective effect of the ultra-low dose of
THC, as well as the induction of cognitive deficits that
lasted for up to 5 months (Tselnicker et al. 2007; Amal et al.
2010), led us to look for long-term biochemical changes in
the brain that may underlie these behavioral outcomes.
Extracellular signal–regulated kinase (ERK) is considered
an important mediator of both preconditioning and post-
conditioning (Gidday 2006; Hausenloy and Yellon 2006;
Hausenloy and Yellon 2009; Pignataro et al. 2009). In our
previous study (Senn et al. 2008) we found the activation of
ERK in the cerebellum 24 h after the injection of an ultra-
low dose of THC to mice. This delayed activation differed
from the previously reported acute effect of the regular high
doses (1–15 mg/kg) of THC that caused a rapid pERK
elevation (10–30 min after injection) which then declined
(Derkinderen et al. 2003; Rubino et al. 2004). In the current
study we found that a single injection of the ultra-low dose
of THC induced long-lasting changes in ERK activity in
various brain regions. Seven weeks after the injection, ERK
activity was elevated in the frontal cortex and the hippo-
campus and suppressed in the cerebellum. The modulation
of ERK activity coincided with a parallel modulation of its
activating enzyme MEK and its inactivating enzyme MKP-
1, indicating the activation of the entire ERK signaling
system in the hippocampus and its suppression in the cer-
ebellum. It was previously suggested that the suppression of
ERK phosphorylation, rather than its stimulation, is related
to the protective effect of cannabinoids against kainite-
induced neuroinflammation (Zhang and Chen 2008). The differential effect of THC on different brain regions
was also manifested by the finding that different down-
stream proteins were activated in different brain regions
7 weeks after the injection of THC. In the hippocampus we
found a significant elevation of phosphorylated cAMP
response element–binding protein (CREB) and no changes
in the level of brain-derived neurotrophic factor (BDNF),
while in the frontal cortex we found no changes in phos-
phorylated CREB, but a significant elevation in BDNF.
Both these proteins were previously found to be acutely
(15–60 min) activated by the conventional (1–15 mg/kg)
doses of THC (Derkinderen et al. 2003; Rubino et al.
2004). Furthermore, both proteins are considered to be
involved in the mechanism of preconditioning. Thus, a
mild ischemic insult used for preconditioning was found to
elevate BDNF in the hippocampus (Truettner et al. 2002)
and to prevent the decline in BDNF that was caused by
lethal ischemia to the brain (Lee et al. 2008). Similarly,
activation of CREB was found in neonatal rat brains when
ischemic preconditioning was introduced to protect against
hypoxic–ischemic brain injury (Lee et al. 2004; Lin et al.
2009a, b). The protective role of these two proteins may be
attributed to their participation in long-term memory for-
mation and synaptic plasticity (Yin and Tully 1996; Lipsky
and Marini 2007; Cunha et al. 2010). The relevance of each
of these proteins to the long-lasting behavioral effects of
the ultra-low dose of THC awaits further research.
In order to further investigate whether the long-term
effect of THC on ERK activity is related to its protective
properties, we searched for possible biochemical inter-
actions between THC and one of the insulting agents
(PTZ). We found that THC and PTZ, when applied sep-
arately, elevated ERK phosphorylation in the hippocam-
pus; hence, we expected to receive an even greater
elevation when applying the two drugs together. How-
ever, when THC was applied 24 h before PTZ, there was
no additive effect; on the contrary, the elevation of pERK
disappeared, and its levels were similar to those in the
control group (Fig. 7). This biochemical interaction
resembled the behavioral interaction, where the cognitive
effects of THC and PTZ failed to appear when both drugs
were applied to the same mice [see Fig. 1 in (Assaf et al.
2011)], and as was also seen with the other insults in the
present study (see Figs. 2, 3).
In summary, we have demonstrated that a single injec-
tion of an ultra-low dose of THC can protect the brain from
different insults that induce cognitive deficits.
When a chronic insult was employed (a daily application
of MDMA), it was necessary to re-administer THC. The
protective effect of THC lasted for many hours and pro-
vided an expanded therapeutic time window. These find-
ings suggest that low doses of THC may be used to protect
the brain not only against acute damage but also against chronic insults or even against neurodegenerative diseases.
The behavioral effects of THC coincided with a long-
lasting modulation of the ERK signaling system and its
downstream proteins BDNF and CREB in different brain
regions. It is suggested that a treatment with such an
extremely low dose of THC has a potential to provide safe,
long-term neuroprotection, without the undesired psycho-
tropic effects of the conventional doses of the cannabinoid
drug, and without inducing downregulation of cannabinoid
receptors that may interfere with the protective effects of
conventional doses of cannabinoid drugs (Zhang and Chen
Acknowledgments Tetrahydrocannabinol was kindly donated by
Prof. R Mechoulam of The Hebrew University of Jerusalem, Israel,
and by The National Institute on Drug Abuse (NIDA), USA. This
study was supported by the Israel Anti Drug and Alcohol Authority

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