Neurochemical Research, Vol. 24, No. 10, 1999, pp.

1233-1240

In-Vitro and In-Vivo Action of Cannabinoids

B. E. Akinshola,1 A. Chakrabarti,2 and E. S. Onaivi2'3

(Accepted May 6, 1999)

The discovery of endocannabinoids such as anandamide and the wide spread localization of
cannabinoid receptors in the brain and peripheral tissues, suggests that the cannabinoid system
represents a previously unrecognized ubiquitous net work in the nervous system, whose physi-
ology and function is unfolding. In this study, we tested the hypothesis that some of the actions
of anandamide are independent of a cannabinoid receptor mechanism. This was accomplished
by the use of cannabinoid agonist and antagonist interaction in an in-vitro and in-vivo test sys-
tems. In-vitro, we used Xenopus laevis oocytes expression system and two-voltage clamp tech-
nique in combination with differential display polymerase chain reaction to determine whether
the differential display of genes following treatment with anandamide may be linked to AMPA
glutamate receptor. The differential expression of genes in vivo after the sub-acute administra-
tion of anandamide could not be directly linked with the AMPA glutamate receptor. In the volt-
age clamp studies we investigated the effects of anandamide on recombinant AMPA GluR3 sub-
unit currents generated by kainic acid in oocytes expressing the AMPA glutamate receptor. In
the in-vitro studies, we present evidence that anandamide inhibited the kainate activated cur-
rents in oocytes expressing AMPA glutamate receptor involves cAMP transduction via a
cannabinoid receptor independent mechanism. In the in-vivo studies, SR141716A, the CB1 an-
tagonist, induced anxiolysis, that was dependent on the mouse strain used in the anxiety model
and blocked the anxiogenic effects of anandamide or methanandamide whereas SR141716A had
no effect on the anandamide inhibition of kainate activated currents in-vitro.

KEY WORDS: Anandamide; AMPA GluR3 receptor subunit; cannabinoid CB1 receptor; cAMP; gene
expression; cannabinoid antagonist; SR141716A; Xenopus oocytes.

INTRODUCTION indicate that cannabinoids may interact with glutamater-

There is increasing evidence that alcohol and
abused drugs, including cannabinoids, alters the expres-
sion of specific genes and affects brain function by inter-
acting with multiple neurotransmitter systems, thereby
modulating the balance between excitatory and inhib-
itory neurotransmitters (1,2). Furthermore recent reports
1
Department of Pharmacology, Howard University College of Med-
icine, Washington DC, 20059.
2
Department of Psychiatry and Pharmacology, Vanderbilt Univer-
sity School of Medicine, Nashville, TN 37232.
3
Address reprint requests to: Dr. Emmanuel S. Onaivi, Department
of Psychiatry (MCN 2213), Vanderbilt University, Nashville, TN
37232., E-mail; Emmanuel.S.Onaivi@Vanderbilt.edu, Phone: 615-
343-0168.
1233
gic NMDA and non-NMDA receptors with implications
in both nervous and immune systems (3-6). For exam-
ple, glutamatergic regulation of cannabinoid receptor
gene expression in the caudateputamen (5) and lipid
mediator, (arachidonic acid) modulation of NMDA re-
ceptor current in oocyte expression system (6) has been
demonstrated. AMPA (a-amino-3-hydroxy-5-methyl-
4-isoxazole-propionic acid) receptors belong to a fam-
ily of ligand-gated glutamate receptors, which include
Abbreviations: AMPA, A-amino-3-hydroxy-5-methyl-4-isoxazole-
propionic acid; NMDA, N-Methyl-D-Aspartate; PCR, polymerase
chain reaction; DDPCR, differential display polymerase chain re-
action; DNA, deoxyribonucleic acid; RNA, ribonucleic acid; KA,
kainate; cAMP, cyclic adenosine monophosphate.
0364-3190/99/1000-1233$ 16.00/0 C 1999 Plenum Publishing Corporation
1234
NMDA (N-methyl-D Aspartate) and KA (Kainate) re-
ceptors. Glutamate is the major excitatory neurotrans-
mitter in the central nervous system, where the major-
ity of fast synaptic transmissions are mediated by
AMPA receptors. Glutamate receptors are important
in synaptic plasticity such as long-term potentiation,
which are implicated in learning and memory func-
tions (7), and also involved in neuronal cell death re-
sulting from neurological diseases, such as epileptic
seizures and Alzheimer's. In addition, following glu-
tamatergic stimulation or under depolarizing condi-
tions, anandamide is released from neural but not glial
culture (8).
The cannabimimetic eicosanoid, anandamide, (ara-
chidonylethanolamide) is an arachidonic acid deriva-
tive, isolated from porcine brain, and characterized as
an endogenous ligand for central cannabinoid CB1 re-
ceptor (9), may represent a novel class of lipid neuro-
transmitters (10). The pharmacological effects of anan-
damide has been the subject of numerous reviews
(10-12). Briefly, in the CNS, anandamide is known to
activate cannabinoid receptors and affect perception
and memory in a similar manner to other cannabi-
noids, indicating a neuromodulatory or neurotransmit-
ter role. Anandamide has also been found to display
cannabinomimetic pharmacological activity in in vivo
and in vitro test systems (4,13). In-vivo cannabinoids
induce a variety of neurobehavioral effects in humans
and animals, some of which can be dissociated by use
of synthetic cannabinoid agonist. We have previously
shown that nabilone and A9-THC share some of the
same behavioral properties while exhibiting opposite
anxiogenic and anxiolytic effects (25). In neuronal cell
lines, anandamide has been reported to inhibit N-type
calcium channels, and Q-type calcium channels, via a
pertussis toxin-sensitive GTP-binding protein pathway,
independent of cAMP metabolism (10,14). However,
cannabinoid inhibition of hormone stimulated adenylyl
cyclase enzyme, also occurs through the GTP-binding
protein. Anandamide inhibited forskolin stimulated
cAMP accumulation in Chinese hamster ovary cells ex-
pressing human cannabinoid receptor, and blocked sea
urchin sperm acrosome reaction, probably, through the
inhibition of ligand-gated ion-channels (10,12,14). Re-
cently, anandamide was reported to play a role in
synaptic plasticity through phosphorylation of a focal
adhesion kinase protein in hippocampal slices and cul-
tured neurons (12).
In the current study, we used Xenopus laevis
oocytes as an expression system and the two-electrode
voltage-clamp technique in combination with differen-
tial display polymerase chain reaction (DDPCR) to
Akinshola, Chakrabarti, and Onaivi
determine whether the differential display of genes fol-
lowing treatment with anandamide may be linked to
AMPA glutamate receptor. In the voltage clamp stud-
ies we investigated the effects of anandamide on re-
combinant AMPA glutamate receptor subunits currents
generated in kainate activated xenopus oocytes express-
ing AMPA GluR3 receptor subunit and whether the
effects of anandamide can be blocked by the CB1 an-
tagonist. In the in-vivo experiments, the effects of the
CB1 antagonist on the anxiogenic effects of anan-
damide/ methanadamide was determined. We present
evidence that anandamide inhibition of AMPA GluR3
receptors expressed in the oocytes could not be blocked
by the CB 1 antagonist but involves a cAMP dependent
mechanism. However, the anxiogenic effects of anan-
damide was blocked by the CB1 antagonist, SRI 4-
1716A. A preliminary report of this study has appeared
in abstract form (3 and 26).
EXPERIMENTAL PROCEDURE
Animals. Male ICR mice( 18-25 gm) housed in groups of five
were used. Mice were injected in different groups with the same
doses of anandamide and cocaine 1.0 and 10 mg/kg ip for 14 days.
At the end of the treatment period the 10.0 mg/kg group were sacri-
ficed and the brains harvested for differential display polymerase
chain reaction procedure. All experiments were approved by the In-
stitutional animal care and use committee (IACUC) of Vanderbilt
University, Nashville, TN USA. All efforts were made to minimize
animal suffering and the number of animals used.
Neurobehavioral Testing. The effect of SR141716A (0.3-
3.0 mg/kg) in different groups of male three mouse lines, C57BL/
6, DBA/2, and ICR in the two compartment black and white box
model of anxiety was evaluated. The two compartment black and
white box was open - topped, with two-fifths painted black and illu-
minated by red light and partitioned from the remainder of the box
which was painted white and brightly illuminated. The compartments
were connected by an opening located at the floor level in the center
of the partition. We have extensively used this model (27) for the
analysis of anxiogenic and anxiolytic agents. Only the ICR mice was
used for the in-vivo antagonist study. In a preliminary study anan-
damide and the more stable CB1 agonist methanandamide induce
anxiogenic profile like THC. Therefore, the ability of SR141716A
to antagonize the acute anxiogenic effects of methanandamide was
determined.
Isolation of Oocytes. Mature Xenopus laevis frogs were ob-
tained from Xenopus I, Ann Arbor, MI and kept in dechlorinated
water tanks at 19-21°C, with a 12-hr light/dark cycle, fed beef liver
at least twice a week. Frogs were anesthetized by submersion in 0.2%
3-aminobenzoic acid ethyl ester (Sigma Chemical, St. Louis, MO),
and a group of oocytes were surgically excised. The oocytes were
separated and the follicular cell layer was removed by treatment with
type I collagenase (Boehringer Mannheim, Indianapolis, IN) for 2 hr
at room temperature.
AMPA GluR3 Receptor Expression. Subunit-specific comple-
mentary RNA (5' capped) for the AMPA GluR3 receptor was synthe-
sized in vitro from Xho 1 linearized template cDNA with T3 RNA
In-Vitro and In-Vivo Action of Cannabinoids
polymerase from mCAP capping kit from Stratagene (La Jolla, CA).
Each oocyte was injected with 16-20 ng of cRNA using a micro-
injector and prepared for electrophysiological recordings. Methods for
oocyte preparation and maintenance are previously described (4,15).
The oocytes were excised from mature female Xenopus laevis frogs
(Xenopus I, Ann Arbor, MI) and manually separated into clusters of
5-10 oocytes. Clusters were placed in a calcium free modified Barth's
solution (MBS) containing (in mM): 88 NaCl, 1 KC1, 2.4 NaHCO3,
0.8 MgSO4 and 10 HEPES (pH 7.5). The follicular cell layer was re-
moved by incubating oocyte clusters in two changes of 2 mg/ml MBS
solution of collagenase A (Boehringer Mannheim, Indianapolis, IN)
with shaking for 1 hour each. Healthy stage VI and V oocytes were
incubated for 3-7 days at 19-2 o C in MBS supplemented with 2 mM
sodium pyruvate, 10,000)M/1 penicillin, 10 mg/l streptomycin, 50 mg/1
gentamycin, 0.5 mM theophyline and 0.9 mM CaCl2 after injection.
Xenopus Oocyte Electrophysiological Recording. The GluR3
receptor expression in the Xenopus oocytes were detected from day
3 for expression of the receptor by the application of 200 MM kainic
acid. After detecting receptor expression, oocytes were placed in a
100 M1 recording chamber perfused with modified Barth's solution
and voltage-clamped using two microelectrodes at a holding poten-
tial of -70 mV. Currents were activated using 200 (MM kainic acid.
Drugs were either coapplied or preapplied for about 30-50 secs,
with a period of about 5 mins between applications. The oocyte re-
sponse to 20 s of superfusion with the 200 MM kainic acid was quan-
tified as the difference between the baseline current and the steady
current observed during superfusion.
Differential Display Polymerase Chain Reaction (DDPCR).
Total RNA was isolated from the brain tissue using TRI REAGENT
(Molecular Research Center Inc). DDPCR was accomplished with
this RNA using the kit from GenHunter, Nashville, TN U.S.A.
Briefly, The differential display methodology was based on a series
of steps including, the isolation of undegraded cellular RNA; re-
verse transcription of the RNA using primers to produce single
stranded DNA complements of a subset of the mRNA; rounds of
PCRs using the primers to amplify the set of cDNAs corresponding
to a smaller subset of the mRNAs; display of the cDNAs produced
in the form of bands on the matrix gel such as the sequencing gel
shown in figure 3A. Bands of interest, such as the ones produced
by anandamide and/or cocaine treatment was excised from the gel,
its cDNA re-amplified by PCR, cloned and then sequenced.
Cloning. Sequencing and Northern Analysis. Differentially ex-
pressed cDNAs were excised from dried gels and re-amplified using
anchored primers HT11G and arbitrary primers. The re-amplified
bands were cloned in pCR-TRAP vector (GenHunter, Nashville, TN,
U.S.A). The inserts were amplified from the plasmids isolated from
these clones and sequenced. The sequencing was done by dideoxy
chain termination method using fluorescent dye in an auto DNA se-
quencer (ABI-377), at the Vanderbilt University Cancer sequencing
center. The amplified inserts were labeled and used as probes for the
Northern analysis as we have previously described (3).
Source of reagents. Anandamide, WIN 55,212-2 mesylate, R(+)-
[2,3-Dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[l,2,3-de]-
l,4-benzoxacin-yl]-(l-naphthalenyl)methanone mesylate; forskolin;
MD-HCL, kainate and cocaine were purchased from Research Bio-
chemicals international, Natick MA.
Data Analysis. Statistical analysis of concentration-response
data was performed using the nonlinear curve-fitting program ALL-
FIT (16). Data were fitted to the logistic equation
y = ((E max - Emin)/(l + (X/IC50)n)) + E min , where x and y are
concentration and responses, respectively, and Emin and Emax are the
minimal and maximal responses, IC50 is the half-maximal inhibitory
1235
concentration and n is the slope factor (pseudo Hill coefficient). In
these studies, Emin and Emax were constrained to 0 and 100 percent
inhibition, respectively. Data were also statistically compared using
the paired t-test, or Instat program as noted. Average values are ex-
pressed as mean ± standard error (SEM). The behavioral data was
analyzed using ANOVA followed by Fisher PLSD and the Sheffe
F-test and when applicable by Dunnett's t-test for multiple compar-
ison with vehicle treated controls.
RESULTS
Cannabinoid Receptor Independent Action of
Anandamide on Kainate Activated AMPA GluRS Cur-
rents. The direct effects of anandamide and its speci-
ficity on kainate-activated currents in the xenopus
oocytes expressing the AMPA GluR3 receptors is
shown in Fig. 1A and 1B. We have previously charac-
terized the effects of anandamide on kainate activated
currents in oocytes expressing homomeric and het-
eromeric recombinant AMPA receptor subunits and
reported that the differential sensitivities of anan-
damide inhibition of the kainate activated currents was
GLuR3 > GluRl/3 > GluRl > GluR2/3 (3). Since
GluR3 subunit was the most sensitive to anandamide
inhibition it was used to characterize the AMPA re-
ceptor currents following activation by kainate. The
modulation of kainate activated AMPA receptor cur-
rents by arachidonic acid and a synthetic cannabinoid
agonist, WIN 55, 212-2 were examined and compared
to that of anandamide. We found that arachidonic acid
(1-300 M) or WIN 55, 212-2 (1-200 M) had little or
no effects on the kainate activated currents in oocytes
expressing AMPA receptors indicating a selectivity of
anandamide actions for the AMPA receptors which
may be independent of cannabinoid receptor activity.
This was confirmed by the cannabinoid receptor (CB1)
antagonist which had no effect either alone or on the
anandamide inhibition of the kainate activated AMPA
GluR3 subunit mediated currents in the oocytes (data
not shown).
Modulation of AMPA-Mediated Currents by Anan-
damide is cAMP Dependent. The involvement of cAMP
transduction mechanism(s) in the anandamide inhibi-
tion of kainate-activated currents in the xenopus
oocytes expressing AMPA GluR3 receptor subunit
was examined and shown in Fig. 2A and 2B. This
was accomplished by evaluating the influence of pre-
incubation with, forskolin, an activator of adenylyl
cyclase, or MDL-HC1, an adenylyl cyclase inhibitor,
on anandamide inhibition of kainate-activated cur-
rents in oocytes expressing the AMPA GluR3 recep-
tor subunit. We report that the effects of anandamide
1236
Fig. 1. Current traces and specificity of anandamide inhibition of
kainate-activated currents in Xenopus oocytes expressing homomeric
AMPA GluR3 receptors. (A) Current traces showing the effect of
100 uM anandamide on currents activated by 200 uM kainic acid in
oocytes expressing AMPA glutamate receptor. (B) The concentration-
response curves of anandamide, arachidonic acid, and WIN55, 212-2
were plotted as percent inhibition of AMPA glutamate receptor
mediated currents. Data points are mean ± SE of 6–8 oocytes.
was potentiated by forskolin whereas MDL-HCl re-
versed the anandamide inhibition of the kainate me-
diated AMPA GluR3 receptor currents in the xenopus
oocytes.
Differential Gene Expression after Treatment of
Mice with Anandamide or Cocaine. The differential
display of genes in the mouse brain following treat-
ment with anandamide, vehicle or cocaine is shown
in Fig. 3A. Northern analysis showing the differential
expression is shown in Fig. 3B, with the 28s and 18s
rRNA at the bottom as a loading control. The probe
used was made from the differentially expressed se-
quence tag shown in table indicates that anandamide
treatment indeed is involved in gene regulation in
Akinshola, Chakrabarti, and Onaivi
Fig. 2. Potentiation of kainate induced currents and time course of
forskolin and MDL-HCl effects in Xenopus oocytes expressing
AMPA receptors. (A) Current traces for anandamide inhibition of
kainate activated AMPA glutamate currents. Preincubation was with
1 uM forskolin and currents activated by 200 uM kainic acid before
the application of anandamide. (B) The percentage of kainate-
activated current is plotted as a function of the time of incubation
with luM forskolin or 10 uM MDL-HCl. Currents were activated by
with 200 uM kainic acid in the presence of 100 uM anandamide.
Data points are a mean ± SE of 6—8 oocytes. Error bars not visible
are smaller than the symbols.
the brain. We have continued to characterize these
genes and also to determine whether the differential
display of genes following treatment with anandamide
may be linked to AMPA glutamate receptor. The dif-
ferentially expressed sequence tags used as a probe for
the Northern analysis has been sequenced. The se-
quence of this EST is shown in Fig. 3C, and may not
be related to AMPA glutamate receptor from the data
bank searches. Cocaine was used as another abused
drug but was not the subject of this investigation. Cu-
riously however, anandamide and cocaine activated a
similar gene Fig. 3A, which was not associated with
the AMPA glutamate receptor gene.
In-Vitro and In-Vivo Action of Cannabinoids
Fig. 3. Differential gene expression after treatment of mice with
anandamide 10 mg/kg or cocaine 10 mg/kg with reference to vehicle
treated controls. (A) The differential display of mRNA derived from
mouse brain following treatment with anandamide, lane 1, vehicle,
lane 2 and cocaine, lane 3. Differential display was undertaken using
an RNA image kit. Arrows indicate putative differentially expressed
genes (cDNAs). (B) Northern analysis showing differential expres-
sion in anandamide treated mice, lane 1, control, lane 2 and cocaine,
lane 3. The 28s and 18s rRNA are shown at the bottom as a loading
control. (C) The sequence tag of differentially expressed gene regu-
lated in animals treated with 10 mg/kg anandamide. The amplified
insert was used as probe for the Northern analysis as shown in B
after labeling.
Antagonism of the Anxiogenic Effects of Methanan-
damide by SR141716A. SR141716A induced anxi-
olytic and anxiogenic profile of response in the mouse
two compartment black and white box model of anxi-
ety which was dependent on the mouse strain Fig. 4.
The anxiolytic profile was characterized by an in-
creased and a decreased exploration in the white and
black chambers respectively in the ICR and C57BL/
6 mice whereas the anxiogenic profile in the DBA/
2 mice was characterized by a decrease and an increase
in exploration in the white and black chambers of the
box. In the ICR mice the acute anxiogenic effects of
methanandamide was antagonized by SR141716A
Fig. 5.
DISCUSSION
Anandamide is an endocannabinoid that has been
shown to have cannabinoid dependent and cannabinoid
independent effects (4). The results obtained from the
1237
present study demonstrates that anandamide inhibits
kainate activated currents in oocytes expressing AMPA
glutamate receptor. This action of anandamide in the
Xenopus oocyte model appears to be cannabinoid re-
ceptor independent and is in agreement with the notion
that anandamide acts on multiple pathways, some of
which are cannabinoid independent (4). It is unlikely
that the Xenopus oocytes express cannabinoid recep-
tors especially as the cannabinoid receptor agonist,
WIN55212-2, and antagonist SR 141716 did not aug-
ment or block the anandamide inhibition of the kainate
activated AMPA glutamate receptor mediated currents
in the oocyte system. Although anandamide has been
shown to mimic a number of the pharmacological ef-
fects of marijuana cannabinoids (13), the data from
the present study provide additional support for canna-
binoid independent effects of anandamide. This im-
plies that the oocyte system expressing AMPA gluta-
mate receptors does not respond via cannabinoid
receptor activation and thus it can be surmised that
anandamide was acting via a cannabinoid independent
mechanism. This observation is also in agreement with
studies that have shown that higher doses of canna-
binoid agonist in in vitro cultured cell systems ex-
pressing CB1 receptors, stimulate the release of arachi-
donic acid, the inhibition of arachidonic re-acylation,
and release of cytoplasmic calcium stores independ-
ent of the cannabinoid receptors (10,17-19). Although
anandamide has been analyzed in human brain (20), its
hydrophobic nature and its ability to pass through
plasma membranes readily, makes it different from
other classical neurotransmitters that are stored in
synaptic vesicles (10). Other cannabinoid independent
effects have been observed for anandamide including
cardiovascular pressor effects and inhibition of astro-
cytic gap junction permeability (21,22). While the con-
centration of anandamide used in this study might
seem slightly higher for physiological relevance, oth-
ers have used carrier protein such as BSA or cyto-
chrome C to deliver anandamide to their site. This may
help explain the apparent low potency of anandamide
in the oocyte experiments. The oocyte has two hydro-
phobic membranes and a large intracellular yolk com-
partment, all of which tend to trap hydrophobic mole-
cules, making anandamide action more diffuse.
Anandamide induced differential regulation of
certain genes in the mouse CNS lends more support
to the observed non-cannabinoid mediated actions of
anandamide as the genes activated or inhibited do not
appear to be AMPA glutamate receptor genes. The
mode of anandamide actions remains unclear as it is
not discernable whether it is preventing transcription
1238
Fig. 4. The effect of SR141716A in the two compartment black and white box model of anxiety. The acute effects was screened 30 mins, after
the administrtation of vehicle and the SR compound in the three mouse lines. Data represents the means ± SEM of the exploratory activity and
the time spent in the black and white chambers respectively in a 5 mins test period (n = 10/group) +/* significantly different from vehicle
animals at p < 0.05 and post hoc analysis by Dunnett's t test.
Fig. 5. The influence of pretreatment with the antagonist SR141716A
on the aversion provoked by methanandamide alone (solid lines)
to the white chamber of the two compartment box in ICR mice.
The 10-min pretreatment with methanandamide in combination
with the SR compound 30 min before 5-min test is shown by
broken lines. *Significantly different from the corresponding vehicle
treated controls at p < 0.05 (ANOVA) and post hoc analysis by
Dunnett's t test.
Akinshola, Chakrabarti, and Onaivi
or translation of genetic messages. However in a re-
cent study we demonstrated that anandamide differen-
tially regulate the expression of cannabinoid CB1 re-
ceptor gene in different mouse strains (13). Therefore
it is unknown at this time, if any of the anandamide in-
duced gene regulation in vivo might be related to the
AMPA glutamate receptor or other genes encoding
other glutamatergic receptors. We have observed that
anandamide and cocaine activate a gene that was not
seen in vehicle treated controls. While this gene was
not AMPA glutamate receptor gene, it is currently
being characterized.
Although the inhibition of kainate activated cur-
rents by anandamide in oocytes expressing AMPA
glutamate receptors appears to be due to cannabinoid
independent mechanism(s), this inhibition may in-
volve a cyclic AMP dependent mechanism independ-
ent of a cannabinoid receptor mediated event. Both
cannabinoid receptors, CB1 and CB2 are known to
couple directly to Gi/0, G proteins and mediate inhibi-
tion or activation of adenylyl cyclase upon binding of
a cannabinoid agonist (23). The results from the pres-
ent study demonstrates the potentiation of anandamide
inhibition of AMPA receptor current by forskolin and
a reversal by an adenylyl cyclase enzyme inhibitor,
MDL-HCl. Whilst this observation may indicate that
In-Vitro and In-Vivo Action of Cannabinoids
the inhibition of the AMPA receptor channels may
involve cAMP, the types of adenylate isozymes in-
volved is currently a matter of speculation. This is
because it was recently reported that cannabinoid re-
ceptor activation differentially regulates the various
adenylyl cyclase (23). Furthermore, AMPA gluta-
mate receptor activation has been linked to cAMP
flux in Xenopus oocytes (10) and to cyclic AMP de-
pendent protein kinase phosphorylation (24). Since
genes encoding nine distinct isoforms of adenylate
cyclase have been identified recently (23), the nature
and the types of adenylate cylase isoforms involved
with the anandamide inhibition of the AMPA gluta-
mate receptor currents remains to be determined. The
in-vivo data obtained indicate that the cannabinoid
(CB1) receptor antagonism induces anxiolysis. This
is in agreement with our previous study (25) that
showed that A9-THC provoked anxiety that may in-
volve the GABA/benzodiazepine supramolecular com-
plex. It is therefore not surprising that in-vivo, the CB1
antagonist blocked anandamide or methanandamide
induced anxiogenesis whereas SR141716A had no ef-
fect on the anandamide inhibition of kainate currents
in the in-vitro oocytes preparation. This is predictable
as the oocytes do not have cannabinoid receptors to
our knowledge.
CONCLUSION
Taken together, the Xenopus oocytes model is an
in vitro system which provides a cellular electrophysio-
logical basis for the elucidation of some of the central
actions of cannabinoids when combined with in vivo
studies. It is therefore concluded that anandamide in-
fluences AMPA glutamate receptor currents in the
oocyte model. This inhibitory influence of anandamide
on AMPA glutamate receptor current involves a cAMP
transduction, and independent of a cannabinoid recep-
tor mechanism. In the in-vivo studies, SR141716A, the
CB1 antagonist, induced anxiolysis, that was depend-
ent on the mouse strain used and blocked the anx-
iogenic effects of anandamide or methanandamide
whereas SR141716A had no effect on the anandamide
inhibition of kainate activated currents in-vitro. In ad-
dition, the differential expression of genes in vivo fol-
lowing the sub-acute treatment with anandamide could
not be directly linked with AMPA glutamate receptors.
Clearly therefore, further studies are warranted to un-
ravel the biological role(s) of endocannabinoids in the
central and peripheral organ system.
1239
ACKNOWLEDGMENTS
This collaborative multidisciplinary research was conducted
in part at the laboratory of molecular and cellular neurobiology of
NIAAA where B.E.A was a Senior Staff Fellow. B.E.A acknowl-
edges the current support of the Howard University - NIAAA CM1-
ARD program. Part of this work was also supported by NIH-NHLBI
grant K0I HL03319 to E. S. O. The authors thank Jim Boulter, Ph.D.
and Steve Heinemann, Ph.D. for providing GluR receptor subunit
clones, Ms Denise Gates and Dr Rong Zang for technical assistance
and Dr. David Lovinger for valuable comments.
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