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 Previous Article | Next Article 
The Journal of Neuroscience, July 15, 1998, 18(14):5322-5332
Hippocampal Neurotoxicity of
 9-Tetrahydrocannabinol
Guy Chiu-Kai
Chan,
Thomas R.
Hinds,
Soren
Impey, and
Daniel
R.
Storm
Department of Pharmacology, University of Washington, Seattle,
Washington 98195
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ABSTRACT |
Marijuana consumption elicits diverse physiological and
psychological effects in humans, including memory loss. Here we report that  9-tetrahydrocannabinol (THC), the major
psychoactive component of marijuana, is toxic for hippocampal neurons.
Treatment of cultured neurons or hippocampal slices with THC caused
shrinkage of neuronal cell bodies and nuclei as well as genomic DNA
strand breaks, hallmarks of neuronal apoptosis. Neuron death induced by
THC was inhibited by nonsteroidal anti-inflammatory drugs, including
indomethacin and aspirin, as well as vitamin E and other antioxidants.
Furthermore, treatment of neurons with THC stimulated a significant
increase in the release of arachidonic acid. We hypothesize that THC
neurotoxicity is attributable to activation of the prostanoid synthesis
pathway and generation of free radicals by cyclooxygenase. These data suggest that some of the memory deficits caused by cannabinoids may be
caused by THC neurotoxicity.
Key words:
THC; cannabinoid receptors; CB1; cell death; hippocampal
neurons; arachidonic acid; reactive oxygen species; SR141716A
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INTRODUCTION |
Marijuana has a long history of
consumption in human societies and is the most commonly used illicit
drug today. Although marijuana is legalized in some states of the
United States for therapeutic usage, its clinical use is controversial.
One established therapeutic application of -9-tetrahydrocannabinol
(THC), the major psychoactive component of marijuana, is the treatment
of nausea and vomiting associated with chemotherapy (Jaffe, 1990 ). Other uses of marijuana include appetite stimulation in cancer patients
and abatement of pain (Abood and Martin, 1992; Voth and Schwartz,
1997). The effects of the drug in humans include hallucination, compromised cognition, memory loss, and immunosuppression (Abel, 1970;
Reisine and Brownstein, 1994).
Human marijuana research is limited, and the retrospective nature of
these studies compromise their interpretation. Nevertheless, marijuana
usage disrupts short-term memory, working memory, and attentional
skills (Fletcher et al., 1996), and induces deficits in mathematical
skills, verbal expression, and memory retrieval (Block and Ghoneim,
1993). Long-lasting cannabis-dependent short-term memory deficits
(Schwartz et al., 1989) and residual neuropsychological effects (Pope
and Yurgelun-Todd, 1996) persist even after abstinence.
Administration of cannabis extracts to rodents also causes long-lasting
memory defects. For example, a 6 month oral administration of THC
reduces maze learning with a residual learning defect that persists
(Fehr et al., 1976; Stiglick and Kalant, 1982b, 1983; Stiglick et al.,
1984). Radial arm learning paradigm showed similar impairments
(Stiglick and Kalant, 1982a). Collectively, studies indicate that
chronic exposure to THC impairs cognition and memory (Carlini et al.,
1970; Abel, 1971).
The effects of THC are mediated primarily through cannabinoid CB1
receptor in the brain (Matsuda et al., 1990) or CB2 receptor in
peripheral tissues (Munro et al., 1993). Cannabinoid receptors couple
to several regulatory pathways, including the cAMP signal transduction
system (Howlett, 1995). Because cannabinoid receptors inhibit adenylyl
cyclases (Howlett and Fleming, 1984) and cAMP regulates synaptic
plasticity (Frey et al., 1993; Chavez Noriega and Stevens, 1994;
Weisskopf et al., 1994; Impey et al., 1996), cannabinoids could inhibit
synaptic function. For example, Schaffer collateral long-term
potentiation in the hippocampus is depressed by THC (Nowicky et al.,
1987; Collins et al., 1994). Alternatively, memory loss may be
attributable to cannabinoid neurotoxicity.
Chronic cannabinoid administration to rats causes distinct
morphological changes in the hippocampus indicative of neurotoxicity (Scallet, 1991). THC exposure for 3 months decreases the mean volume of
neurons and their nuclei, the synaptic density, and the dendritic
length of CA3 pyramidal neurons (Scallet et al., 1987). Exposure to THC
also decreases neuronal density in rat hippocampus (Landfield et al.,
1988). Although animal studies suggest that chronic exposure to THC may
be toxic for hippocampal neurons, mechanisms for THC neurotoxicity have
not been defined.
To determine whether THC is toxic for hippocampal neurons and to
explore possible mechanisms, we examined its influence on the viability
and morphology of cultured rat hippocampal neurons and hippocampal
slices. Our data indicate that THC induces cell death in neurons that
may be attributable to activation of the phospholipase
A2-cyclooxygenase pathway.
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MATERIALS AND METHODS |
Primary neuron cultures and measurement of cell
death. Primary hippocampal neurons were established from postnatal
day one Sprague Dawley rat pups and maintained in defined medium with minimal supplements. Pups were killed by decapitation and the hippocampi were digested in 5 ml of 10 U/ml papain at 37°C for 20 min. After two rinses, the tissue was triturated (two rounds, 10-15
pipetting each) in dissociation medium (27 mM
K2S04, 1 mM kynurenic acid,
15 mM MgCl2, 74 mM
Na2SO4, 18 mM glucose, 225 µM CaCl2, 0.0012% phenol red, and 2 mM HEPES, pH 7.4) using a 5 ml disposable plastic pipette.
Ninety-six-well plates were coated with poly-D-lysine (66 µg/ml). Cells were plated into the inner 60 wells of the 96-well
plate (the peripheral wells were filled with water to act as humidity
barrier) at 5 × 104
cells/cm2 and maintained in 200-250 µl of
Neurobasal medium (Life Technologies, Gaithersburg, MD) in the presence
of N-2 supplement (Bottenstein and Sato, 1979) with 10 U/ml penicillin,
10 µg/ml streptomycin, and 0.5 µg/ml glutamine for 10-16 d before
use. Because Neurobasal medium and N-2 did not maintain neuron
viability for extended periods, the media was modified. These
modifications included adding 0.1% chicken ovalbumin (Sigma, St.
Louis, MO) and increasing sodium selenite to at least 300 nM. The amount of selenite added (up to micromolar) was
determined empirically for each batch of N-2. Primary neurons cultured
under these conditions survived 4-5 weeks. Serum-free Neurobasal
medium with supplements has been used extensively for sustaining the
survival of cultured CNS neurons (Brewer et al., 1993; Otsuki et al.,
1994; Takeshima et al., 1994; Morrison et al., 1996; Xiang et al.,
1996). However, supplements with very high contents of antioxidants,
such as B27, were not suitable for our studies (see Results). Neurons
maintained in the presence of serum experience serum withdrawal-induced
cell death at the time of THC treatment. All cannabinoid dilutions were
performed in siliconized tubes. Absolute ethanol was used as carrier or
vehicle for THC. Controls and vehicle-treated controls were obtained
from cells treated with comparable concentration of ethanol as
THC-treated samples. MTT assays were performed as previously described
(Hansen et al., 1989). At the end of the incubation, 25 µl of MTT (5 mg/ml) was added to the cells in 100 µl of growth media. The plates
were incubated for 2 hr at 37°C in a CO2 incubator and
solubilized overnight in 9% SDS and 22% DMF before determination of
absorbance at 570 nm. SR141716A was provided by Research Biochemicals
(Natick, MA) as part of the Chemical Synthesis Program of the National
Institutes of Mental Health, Contract N01MH30003.
Inhibitor experiments were performed with 10-16 d cultured neurons.
Unless otherwise stated, neurons in 96-well plates were pretreated with
the inhibitors for 1 hr before treatment with THC. Various
concentrations of inhibitors were tested for their effect on neuron
viability in the absence of THC to define nontoxic concentrations for
each inhibitor. The inhibitors and concentrations examined included
quinacrine, 0.2-5.0 µM; indomethacin, 1.0-5.0 µM; nordihydroguaiaretic acid (NDGA), 1.0-5.0
µM; EGTA, 1.5 mM; vitamin E, 20-200
µM; Trolox, 100 µM; aspirin, 1.0-5.0
µM; dexamethasone, 100 nM; and actinomycin D,
2.0 µg/ml.
Arachidonic acid release assay. Hippocampal neurons (plated
at 0.6 × 106/well in six-well plates) were
grown in culture for 7-11 d before loading with 0.5 µCi/ml
[5,6,8,9,11,12,14,15-3H(N)]arachidonic
acid (223 Ci/mmol; DuPont NEN, Boston, MA) in a CO2
incubator for 3.5-4.5 hr. The cells were rinsed twice with Neurobasal
medium and treated in 2 ml conditioned media in the presence or absence
of THC. At various time points after treatment with THC, 100 µl of
supernatant was withdrawn and centrifuged at 14,000 × g at 25°C for 5 min. The supernatant was transferred to a
fresh tube for liquid scintillation counting. Data reported are
averages from three independent experiments.
Terminal deoxynucleotidyl transferase-mediated biotinylated UTP
nick end labeling (TUNEL) and bisbenzimide staining of cultured hippocampal neurons. Neurons were cultured on 18 mm circular
coverglass slips (Fisher Scientific, Houston, TX) coated with
poly-D-lysine in 1 ml of growth medium in 12-well plates.
TUNEL labeling was performed as recommended by the manufacturer
(in situ cell death detection kit, POD; Boehringer Mannheim,
Indianapolis, IN). Horseradish peroxidase reactions were performed as
recommended using 3,3'-diaminobenzidine (DAB substrate kit for
peroxidase; Vector Laboratories, Burlingame, CA). Counterstaining was
done using 1% toluidine blue in 1% sodium borate. For bisbenzimide
staining, neurons were fixed with 4% formaldehyde, rinsed with PBS,
and stained with 2.5 µg/ml Hoechst 33342 (Sigma) at 25°C for 5 min.
After rinsing with PBS and mounting on slides with GelMount, the slices
were examined by fluorescence microscopy.
Calcium imaging. Primary hippocampal neurons were maintained
for 8-16 d in four-chambered Lab-Tek coverglass (Nalge Nunc, Roskilde,
Denmark) coated with poly-D-lysine. On the day of
Ca2+ imaging, the conditioned media from the neurons
were removed and saved. Cells were rinsed with 1 ml of imaging buffer
and loaded with 1.5 µM fura-2 AM (Molecular Probes,
Eugene, OR) in the same buffer in the dark for 20 min at 25°C. After
rinsing with 1 ml imaging buffer, the conditioned media were added back
to the cells, and the coverglass was incubated in the CO2
incubator until use (within 2-4 hr). Calcium imaging was performed and
interpreted as described (Wayman et al., 1995). Unless otherwise
stated, stock THC and other reagents were first added to the buffer and
then to hippocampal neurons incubating in the same volume of the
identical buffer. Final concentrations were 10 µM THC, 5 µM SR141716A, and 4 mM LaCl3.
THC treatment of rat hippocampal slices. Brain transverse
sections (400 µm) were cut from female adult (~250 gm) Long-Evans rats that were killed by decapitation. Hippocampal slices were perfused
in continuously oxygenated artificial CSF (ACSF; in mM: 120 NaCl, 3.5 KCl, 1.3 MgCl2, 2.5 CaCl2, 1.25 NaH2PO4,
25.6 NaHCO3, and 10 glucose) at 30°C for 1 hr
before adding THC (Research Biochemicals). Stock solutions of THC were
dissolved in 100% ethanol, and the final ethanol concentration after
dilution was 0.01%. After 2 hr of perfusion with THC (flow rate, 75 ml/hr) and 3 additional hr with ACSF alone, the sections were fixed
overnight in 4% formaldehyde and then cryostatically protected in 30%
sucrose. Forty µm (for propidium iodide staining) or 25 µm (for
TUNEL assay) sections were then made using a microtome. For propidium
iodide staining, sections were treated with 5 ml of 10 µg/ml RNase A
at 25°C for 30 min and stained with 1-2 ml of 0.5 µg/ml propidium
iodide at 25°C for 5 min. For TUNEL assays, 25 µm sections were
adhered to charged slides (Superfrost Plus, VWR Scientific) at 25°C
and allowed to dry. Sections were treated with 10 µg/ml proteinase K
in 10 mM Tris, pH 7.4, at 25°C for 30 min, rinsed four
times in 10 ml PBS, permeabilized in 10 ml 1× PBS, 0.1% Triton X-100 at 25°C for 15 min, and then incubated in 250 µl terminal
deoxytransferase (TdT) buffer (Life Technologies) and 4.0 µM biotin-14-dATP (Life Technologies) at 25°C for 10 min. Two hundred fifty microliters (100 U/ml) of a recombinant TdT
(Life Technologies) were added in the same buffer and incubated at
37°C for 90 min. Reactions in small volume were performed in covered
dishes in the CO2 incubator to minimize evaporation. The
reaction was terminated by rinsing slides twice for 10 min with 10 ml
of PBS at 25°C, followed by adding 10 ml of 300 mM NaCl
with 30 mM sodium citrate and incubating at 25°C for 15 min. After rinsing with PBS, the sections were incubated in PBS
containing 2% BSA and 4.0 µg/ml lissamine rhodamine-conjugated streptavidin (Jackson ImmunoResearch, West Grove, PA) at 25°C for 60 min. After rinsing several times with PBS, the slices were subjected to
one more round of amplification with biotinylated anti-streptavidin
antibodies from goat (1 µg/ml; Vector Laboratories) and lissamine
rhodamine-conjugated streptavidin (8.0 µg/ml) before mounting with
GelMount (Biomeda) for confocal microscopy.
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RESULTS |
THC is toxic for cultured hippocampal neurons
To determine whether THC is neurotoxic and to elucidate mechanisms
for toxicity, we examined its effect on the viability of cultured
primary hippocampal neurons from neonatal rats (Fig. 1). Survival of hippocampal neurons was
monitored as a function of THC concentration and time after treatment
using the MTT tetrazolium salt
[3,(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide] assay as a measure of cell viability (Hansen et al., 1989).
Concentrations of THC as low as 0.5 µM were toxic to
hippocampal neurons, and the rate of neuron death increased with THC
concentration (Fig. 1A,B). For
example, 50% of the neurons were killed within 3 hr of treatment with
10 µM THC or 6 d after exposure to 1.0 µM THC. The lower doses used are comparable to THC
concentrations measured in human plasma after consumption of a single
marijuana cigarette (Chiang and Barnett, 1984). THC concentrations in
the brain may be even higher after smoking marijuana, because THC is
lipophilic (Thomas et al., 1990) and readily crosses the blood-brain
barrier. Blood levels of THC reach a maximum shortly after smoking
marijuana and decline rapidly thereafter. However, even a 15 min
exposure to THC was toxic for cultured hippocampal neurons (Fig.
1C). Cultured cortical neurons were much less sensitive to
THC than hippocampal neurons (data not shown). Because THC is
lipophilic, some of its effects may be attributable to interactions
with membrane lipids rather than receptor activation. However,
SR141716A, an antagonist of CB1 receptors (Rinaldi-Carmona et al.,
1994), completely inhibited THC-induced neuron death (Fig.
2). These data indicate that THC toxicity
is mediated through CB1 receptors and not caused by nonspecific interactions with the membrane lipid phase.
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Figure 1.
THC is toxic for cultured hippocampal neurons.
Primary hippocampal neurons were treated with various concentrations of
THC at high doses (A) (in µM):
open squares, 3.5; open triangles, 5.0;
filled circles, 7.5; filled triangles,
10; and filled squares, 20; or low doses
(B) (in µM): open
squares, 0.20; open triangles, 0.38;
filled circles, 0.50; filled triangles,
1.0; and filled squares, 2.0. C, Primary
hippocampal neurons were treated with 3.5 µM THC for the
indicated periods (15, 30, or 45 min or 20 hr), followed by removal of
the drug and reincubation in conditioned media overnight. Cell
viability was measured by the MIT assay. One hundred percent viability
is defined as the MTT conversion by cells treated with the carrier for
the duration of the experiment.
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Figure 2.
THC toxicity requires activation of the CB1
receptor. Neurons were treated with 10 µM THC in the
presence or absence of the CB1 antagonist SR141716A
(SR). Cell viability was analyzed 5 hr after treatment
by the MTT assay. Neurons were preincubated with SR for 20 min before
treating with THC.
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THC-induced neurotoxicity is not attributable to inhibition of
adenylyl cyclases
THC neurotoxicity might be attributable to suppression of adenylyl
cyclase activity, because cannabinoid receptors couple to inhibition of
adenylyl cyclases through Gi (Howlett and Fleming, 1984;
Howlett et al., 1986; Howlett, 1995), and neuron survival depends on
optimal cAMP (Deckwerth and Johnson, 1993; Holcomb et al., 1995; Nobes
and Tolkovsky, 1995; Xia et al., 1995). To evaluate this possibility,
cultured neurons were treated with pertussis toxin (Ptx) to inactivate
Gi and monitored for THC toxicity. Neither Ptx pretreatment
nor forskolin, an adenylyl cyclase activator, protected neurons from
THC-induced death (Fig. 3). This suggests that THC neurotoxicity is not caused by adenylyl cyclase inhibition. Interestingly, actinomycin D, an inhibitor of transcription, blocked THC-induced toxicity (Fig. 3). This suggests that THC may activate a
transcription-dependent cell death program.
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Figure 3.
THC toxicity is not attributable to changes in
cAMP. Hippocampal neuron viability was measured after treatment with 10 µM THC alone (THC), THC with 200 ng/ml
pertussis toxin (Ptx; pretreated for 16 hr), with THC in
the presence of 10 µM forskolin (Fsk), or
with THC in the presence of 2 µg/ml actinomycin D (Act
D). With the exception of Ptx, neurons were treated with
reagents for 5 hr. p values are from Student's
t test: NS, not significant;
**p < 0.005. Data are triplicates from one
representative experiment. Comparable results were obtained in four
independent experiments. Ctrl, Control treatment with
0.01% ethanol, the carrier for THC. Error bars indicate ±SD.
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THC increases intracellular Ca2+ in
hippocampal neurons
Intracellular free Ca2+
([Ca2+]i) is modulated by
activation of cannabinoid receptors (Mackie and Hille, 1992; Mackie et
al., 1993; Shen et al., 1996; Sugiura et al., 1996) and prolonged
increases in [Ca2+]i are generally
toxic (Trump and Berezesky, 1995; McConkey and Orrenius, 1996).
Consequently, the effects of THC on
[Ca2+]i were analyzed by fluorescent
imaging using the Ca2+ fluorophore fura-2 AM (Fig.
4). Addition of 10 µM THC
to hippocampal neurons caused a delayed increase in
[Ca2+]i that reached a maximum at 90 min (Fig. 4A). Treatment of neurons with 2 µM THC also increased
[Ca2+]i but with slower kinetics (data
not shown). When cells were treated with THC without extracellular
Ca2+ and in the presence of EGTA, there was no
significant increase in [Ca2+]i (Fig.
4A). Addition of extracellular
Ca2+ at the end of this incubation led to an
instantaneous rise in [Ca2+]i,
suggesting that a plasma membrane channel is activated by THC.
Lanthanum, a potent blocker of Ca2+ channels,
completely inhibited this rise in
[Ca2+]i (Fig. 4B).
THC-induced Ca2+ increases were also inhibited by
the cannabinoid receptor antagonist SR141716A (Fig. 4C).
Because stimulation of CB1 receptors increased [Ca2+]i, we examined the effect
of chelating Ca2+ on THC neurotoxicity. Chelation of
extracellular Ca2+ with EGTA did not rescue
hippocampal neurons from THC toxicity (Fig.
5). This suggests that THC neurotoxicity
is not dependent on increases in intracellular Ca2+,
because THC-stimulated increases in
[Ca2+]i required extracellular
Ca2+. This distinguishes THC neurotoxicity from
other forms of toxicity that are triggered by increased
[Ca2+]i.
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Figure 4.
THC induces a delayed increase in intracellular
Ca2+ that is dependent on extracellular
Ca2+. [Ca2+]i of
THC-treated primary hippocampal neurons was monitored by fura-2
fluorescent imaging. The calibrated
[Ca2+]i (nM) was plotted
against time (minutes). A, THC was added to neurons in
the presence of 1.5 mM CaCl2 (THC + Ca2+) at the indicated time (arrow).
No increase in intracellular Ca2+ was seen when the
ethanol carrier (0.01%) was added alone (data not shown) or when THC
was added to cells incubated without Ca2+ in the
presence of 5 mM EGTA (THC  Ca2+). Four mM LaCl3
(B) or 5 µM SR141716A
(C) blocked increases in
[Ca2+]i caused by 10 µM
THC.
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Figure 5.
THC neurotoxicity is blocked by inhibitors of the
PLA2 cyclooxygenase pathway and antioxidants, but not by
Ca2+ chelation. Neurons were treated with 0.2%
ethanol (Ctrl), 3.5 µM THC
(THC), or THC in the presence of 0.2 µM
quinacrine (Quin), 1 µM indomethacin
(Indo), 5 µM aspirin (Asp),
5 µM NDGA, 1.5 mM EGTA, or 20 µM  -tocopherol (Vit E). After 20 hr,
neurons were assayed for viability as described in Materials and
Methods. Neurons were preincubated with inhibitors for 60 min before
adding THC. Numbers in parentheses represent the number of independent
experiments, each performed in triplicate. Error bars indicate SD.
p values are from Student's t test.
*p < 0.05; **p < 0.0005; NS,
not significant.
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THC neurotoxicity is abrogated by vitamin E as well as inhibitors
of phospholipase A2 and cyclooxygenase
Activation of cannabinoid receptors can stimulate phospholipase
A2 (PLA2), which catalyzes release of
arachidonic acid (Reichman et al., 1988; Audette et al., 1991;
Shivachar et al., 1996), the substrate for several major pathways. One
branch, catalyzed by cyclooxygenases (COX), generates prostanoids,
prostaglandins, and thromboxanes. Another pathway catalyzed by
lipoxygenases produces leukotrienes. Both pathways generate free
radicals (Wei et al., 1981; Pourcyrous et al., 1990; Yamamoto, 1991;
Lafon-Cazal et al., 1993) that can lead to lipid peroxidation and cell
death. Preincubation with quinacrine, an inhibitor of
PLA2, partially protected neurons from THC-induced
cell death (Fig. 5). Neurons were also protected by dexamethasone (data
not shown), which induces the expression of annexin I (Flowers, 1988).
Annexin I inhibits PLA2 (Croxtall et al., 1995) and the
expression of COX (Fu et al., 1990). Indomethacin and aspirin,
inhibitors of COX, completely protected neurons from THC, whereas NDGA,
a lipoxygenase inhibitor, was not protective (Fig. 5). Because COX
generates reactive oxygen species (ROS), which can cause cell death by
stimulating the oxidation of lipids, proteins, and nucleic acid
(Yamamoto, 1991; Troy and Shelanski, 1994; Greenlund et al., 1995;
Simonian and Coyle, 1996), we examined the effect of antioxidants on
THC-induced neuron death. Vitamin E completely protected neurons from
THC (Fig. 5). These data suggest that THC may kill neurons by
stimulation of the cyclooxygenase pathway with generation of ROS.
THC stimulates the release of arachidonic acid from primary
hippocampal neurons
If THC neurotoxicity is attributable to activation of the
PLA2-COX pathway in neurons, THC should stimulate
arachidonic acid release. Treatment of cultured hippocampal neurons
with THC induced the release of arachidonic acid from cultured
hippocampal neurons, which was completely blocked by SR141716A, the CB1
receptor antagonist (Fig. 6). The
kinetics for arachidonic acid release were dependent on THC
concentration. For example, the rate of arachidonic acid release was
three times faster in the presence of 10 µM THC (Fig. 6B) compared with 3.5 µM THC (Fig.
6A). Interestingly, pertussis toxin did not affect
THC-induced increases in arachidonic acid release, suggesting that the
enhancement of arachidonic acid release by THC is not mediated through
Gi coupling. To determine whether exogenously added
arachidonic acid affects neuron viability, primary hippocampal neurons
were treated with increasing concentrations of arachidonic acid and
monitored for cell viability. Exogenous arachidonic acid at 10 µM killed 100% of the cultured hippocampal neurons 24 hr
after exposure (data not shown). However, it is unlikely that free
arachidonic acid added to the outside surface of a cell is comparable
to arachidonic acid release by activation of PLA2 caused by
receptor stimulation.
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Figure 6.
THC induces arachidonic acid release from
hippocampal neurons. Arachidonic acid release was monitored in cultured
hippocampal neurons after treatment with THC. Hippocampal neurons were
maintained in culture for 7-8 d and loaded with
[3H]arachidonic acid for 3-4 hr and treated as
described in Materials and Methods with 3.5 µM
(A) or 10 µM
(B) THC. SR141716A (SR)
pretreatment (5 µM) was done 5 min before THC treatment.
Pertussis toxin (Ptx) was added at 300 ng/ml, 12 hr
before the addition of THC. Error bars indicate SD.
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THC induces cell body shrinkage, nuclear condensation, and genomic
DNA strand breakage in cultured hippocampal neurons
To determine whether THC induces morphological changes in cultured
hippocampal neurons, we examined its effects on cell morphology and
genomic DNA integrity using TUNEL for in situ DNA labeling (Gavrieli et al., 1992). Neuron cell bodies were lightly counterstained (Fig. 7A), whereas DNA strand
breaks were detectable as small dark bodies in culture (Fig.
7B,C). THC increased DNA strand
breaks that were evident within days after treatment with 2 µM THC (Fig. 7B) or within 6 hr after
treatment with 10 µM THC (Fig. 7C). In addition, THC caused a marked decrease in cell body size relative to
control cells.
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Figure 7.
THC induces nuclear shrinkage and genomic DNA
breakage in primary hippocampal neurons. Neurons were maintained for
10 d before treatment with 0.01% ethanol for 3 d
(A, D), 2.0 µM THC for
3 d (B, E), or 10 µM
THC for 6 hr (C, F). Cells were
analyzed using the TUNEL assay (A-C)
coupled to horseradish peroxidase assay or stained with 2.5 µg/ml of
Hoechst 33342 (D-F) as described
in Materials and Methods. Light microscopic images of horseradish
peroxidase staining and counter staining with 0.1% toluidine blue
(A-C) and fluorescent images
(D-F) are shown. Thin
arrows indicate representative examples of condensed chromatin;
thick arrows indicate normal chromatin. Scale bars, 50 µm.
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Neurons were also stained with Hoechst 33342, a cell-permeant
bisbenzimide DNA dye, to examine the effects of THC on nuclear morphology. Treatment of neurons with 2 µM (Fig.
7E) or 10 µM THC (Fig. 7F)
increased the percentage of cells exhibiting nuclear condensation
(50.4%, n = 241; and 56.5%, n = 250, respectively) compared with neurons treated with carrier alone (Fig.
7D; 13.6%, n = 301).
THC treatment causes nuclear contraction and genomic DNA breakage
in hippocampal slices
Rat hippocampal slices were exposed to THC, fixed, and treated
with RNase A before staining with propidium iodide to stain DNA or with
terminal deoxytransferase for TUNEL assay. The CA1 pyramidal cell layer
of hippocampal slices was then examined by confocal microscopy (Fig.
8). In control slices, nuclear propidium iodide staining in the CA1 pyramidal cell layer was diffuse and relatively uniform (Fig. 8A, m.). In
contrast, nuclei in slices treated with 2.5 µM THC were
condensed and contracted compared with controls (Fig.
8B, m.). An analysis at higher
magnification indicated that the nuclei of THC-treated neurons were
~50% smaller (Fig. 8D) than untreated nuclei (Fig.
8C). However, the distance between neighboring nuclei and
the size of nucleoli were unaffected.
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Figure 8.
THC induces nuclear shrinkage and DNA cleavage in
neurons of hippocampal slices. Hippocampal slices perfused with 0.01%
ethanol, the carrier for THC, (A, C,
E, G) or with 2.5 µM THC
(B, D, F, H) for 2 hr followed by 3 hr of perfusion in buffer alone. Slices were stained
with propidium iodide (A-D) or subjected
to TUNEL assays (E-H) as
described in Materials and Methods. Confocal microscopic images of the
CA1 pyramidal cell layer are shown. s.o.,
Stratum oriens; m., CA1 pyramidal cell layer;
s.r., stratum radiatum of the
hippocampus. Scale bars: A, B, 50 µm;
C, D, 10 µm; E,
F, 100 µm; and G, H, 25 µm.
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TUNEL staining of CA1 pyramidal neurons in hippocampal slices was
markedly increased by THC (Figs.
8F,H) compared with control slices (Figs. 8E,G). Although other
areas of the hippocampus, including area CA3 and the dentate gyrus,
were also affected by THC, the increase in TUNEL staining was most
evident in the CA1 pyramidal cell layer. These data indicate that CA1
pyramidal neurons are sensitive to THC in tissue slices where many
normal cellular interactions are preserved.
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DISCUSSION |
Although marijuana is known to cause memory deficits in
humans and laboratory animals, the mechanism(s) for these effects have
not been elucidated. Because administration of marijuana and
cannabinoids to rodents causes a reduction in neuron density in the
hippocampus (Landfield et al., 1988), deficiencies in memory might be
attributable to neurotoxicity. The objectives of this study were to
determine whether THC, a major psychoactive component of marijuana, is
toxic for hippocampal neurons and to characterize mechanisms for
neurotoxicity. We examined THC neurotoxicity in vitro using
cultured hippocampal neurons and hippocampal slices to identify signal
transduction pathways contributing to toxicity.
On the basis of the data presented in this study, we conclude that
binding of THC to cannabinoid CB1 receptors in hippocampal neurons
leads to neuronal death. THC is neurotoxic at concentrations as low as
0.5-1.0 µM, which are comparable to THC levels measured in human plasma after consumption of marijuana cigarettes. Although THC
neurotoxicity was unaffected by Ca2+ chelators and
activators of adenylyl cyclase, it was abated by inhibitors of
PLA2 and completely blocked by aspirin and indomethacin, inhibitors of COX. This suggests that activation of PLA2 by
THC may contribute to increases in arachidonic acid. Because protection by quinacrine was only partial, other mechanisms may contribute to the
arachidonic acid release. For example, THC may also increase intracellular arachidonic acid by inhibition of arachidonic acid acylation (Reichman et al., 1991). We hypothesize that THC-induced neuron death is triggered by a signal transduction cascade that increases arachidonic acid and activates COX with the formation of ROS
(Fig. 9). THC enhancement of arachidonic
acid release from cultured neurons and protection of neurons from THC
by vitamin E supports this hypothesis.
View larger version (23K):
[in this window]
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|
Figure 9.
Signaling pathways contributing to THC-induced
neuronal death. It is hypothesized that excessive stimulation of
cannabinoid receptors (CB1) by THC stimulates the
production of arachidonic acid (AA) by several pathways.
Cyclooxygenase (COX) catalyzes the formation of
prostaglandins (PGs), thromboxanes (TXs),
and reactive oxygen species (ROS), which stimulates
peroxidation of lipids, proteins, and DNA. In addition,
cannabinoid-induced transcriptional events may also contribute to THC
induction of neuron death.
|
|
Our data implicating increases in arachidonic acid and activation of
COX in THC neurotoxicity are consistent with other reports in the
literature that cannabinoids stimulate arachidonic acid release from
cultured astrocytes (Shivachar et al., 1996), cortical slices (Reichman
et al., 1987), and lymphocytes (Audette et al., 1991). Furthermore,
cannabinoids have been reported to stimulate cyclooxygenases (Reichman
et al., 1987). The release of arachidonic acid and the subsequent
generation of ROS in other systems are both implicated in cell damage
(Kuehl and Egan, 1980) and cell death (Tang et al., 1996; Chen et al.,
1997; Li et al., 1997; Wissing et al., 1997). ROS oxidizes membrane
lipids, nucleic acids, and proteins, all of which can lead to necrotic
or apoptotic cell death. Interestingly, THC causes DNA strand breaks,
nuclear contraction, and cell body shrinkage, which are characteristic
of cells undergoing apoptosis (Hockenbery, 1995; Majno and Joris,
1995).
Data in this paper suggest that stimulation of transcription may be
important for THC neurotoxicity. Although the transcriptional events
necessary for THC neurotoxicity were not defined, there are several
possibilities. For example, activation of cannabinoid receptors induces
the expression of immediate early genes Krox24, Krox20, and Jun B
(Bouaboula et al., 1995). It also increases nuclear factor- B
activity (Daaka et al., 1997), which activates the expression of
several genes including tumor necrosis factor (TNF) and other
cytokines (for review, see Baeuerle and Baltimore, 1996).
Interestingly, TNF induces apoptotic cell death. Coactivation of
these pathways with THC stimulation of the arachidonic acid release and
generation of ROS may be required for THC neurotoxicity. Because
inhibitors of protein synthesis induce the expression of Bcl-2 and
activate antioxidant pathways (Furukawa et al., 1997), inhibitors of
transcription may have similar effects. Consequently, the generation of
ROS by THC may be antagonized by induction of Bcl-2, superoxide
dismutases, and catalase.
The pharmacology of cannabinoids is characterized by a multiplicity of
effects in humans and rodents. Treatment of rats and other laboratory
animals with THC elicits several responses, including decreases in
memory, antinociception, hypothermia, catalepsy, and diminished
spontaneous motion (for review, see Dewey, 1986). The hippocampal
complex is pivotal for memory in humans and laboratory animals.
Hippocampal lesions in humans (Scoville and Milner, 1957; Damasio et
al., 1985) or rodents (Olton, 1987) decrease the ability to acquire new
information or recover previously learned knowledge. Intrahippocampal
injection of cannabinoids in rats impairs spatial memory, suggesting
that memory defects are mediated primarily by cannabinoid receptors in
the hippocampus (Lichtman et al., 1995). The data reported in our study
are consistent with this hypothesis and suggest that marijuana-induced
memory defects in rodents may be attributable to THC neurotoxicity.
Also consistent with this hypothesis is the expression of high levels
of cannabinoid receptors in the hippocampus (Herkenham et al.,
1991).
Marijuana is used therapeutically for several conditions, including
treatment of pain and inflammation, nausea caused by cancer chemotherapy, and muscle spasms associated with multiple sclerosis (for
review, see Hollister, 1986). However, clinical use of cannabinoids may
be compromised by their disruptive effects on information processing
and storage. Although there is no direct evidence that marijuana is
neurotoxic for human brain, some of the memory deficits associated with
its use may be caused by neuronal death in the hippocampus.
Consequently, memory defects caused by marijuana might be reduced by
aspirin, indomethacin, or vitamin E. Interestingly, clinical studies
indicate that elevation of prostaglandins caused by marijuana smoking
can be suppressed by indomethacin (Perez Reyes et al., 1991). This
indicates that marijuana use in humans activates the
PLA2-COX pathway and suggests that indomethacin or aspirin
might be appropriate for protection against cannabinoids. Although
speculative, this hypothesis is based on an analysis of the molecular
mechanisms underlying THC neurotoxicity. The identification of
pharmacological methods to prevent marijuana neurotoxicity may be an
important consideration when using marijuana medicinally.
In summary, treatment of hippocampal neurons with THC induces
transcriptionally dependent cell death. This suggests that memory loss
associated with marijuana treatment of rodents, and perhaps humans, may
be attributable to THC neurotoxicity. The identification of a specific
signal transduction system responsible for THC toxicity and the
discovery that aspirin and vitamin E inhibit this neurotoxicity suggest
pharmacological tools to block THC-induced cell death. In addition,
endogenous ligands for cannabinoid receptors have been identified
(Devane et al., 1992; Priller et al., 1995). Cannabinoid receptors may
play an important role during neural development by mediating cell
death in specific populations of neurons in response to the local
release of endogenous cannabinoids such as anandamide.
| |
FOOTNOTES |
Received Feb. 2, 1998; revised April 18, 1998; accepted April 30, 1998.
This research was supported by National Institutes of Health Grant
NS20598 and the Alcohol and Drug Abuse Institute, University of
Washington. Dr. G. C. Chan was supported by postdoctoral training Grants DE07023-21 and DA07278-01A1. We thank Lisa Prichard, Scott Wong,
Mark Nielsen, Jia Wei, Dr. Christoff Deloulme, Dr. Lauren Baker, and
Dr. Enrique Villacres for critical reading of this manuscript.
Correspondence should be addressed to Daniel R. Storm, Department of
Pharmacology, Box 357280, University of Washington, Seattle, WA 98195.
| |
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Copyright © 1998 Society for Neuroscience 0270-6474/98/18145322-11$05.00/0
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Abstract
Introduction
