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The Journal of Neuroscience, January 1, 2001, 21(1):109-116
Localization and Mechanisms of Action of Cannabinoid Receptors at
the Glutamatergic Synapses of the Mouse Nucleus Accumbens
David
Robbe1,
Gérard
Alonso2,
Florence
Duchamp2,
Joël
Bockaert1, and
Olivier J.
Manzoni1
Centre National de la Recherche Scientifique
1 Unité Propre de Recherche 9023 and
2 Unité Mixte de Recherche 5101, 34094 Montpellier
Cedex 05, France
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ABSTRACT |
Despite the role of excitatory transmission to the
nucleus accumbens (NAc) in the actions of most drugs of abuse, the
presence and functions of cannabinoid receptors (CB1) on the
glutamatergic cortical afferents to the NAc have never been explored.
Here, immunohistochemistry has been used to show the localization of CB1 receptors on axonal terminals making contacts with the NAc GABAergic neurons. Electrophysiological techniques in the NAc slice
preparation revealed that cannabimimetics [WIN 55,212,2 (WIN-2) and
CP55940] strongly inhibit stimulus-evoked glutamate-mediated transmission. The inhibitory actions of WIN-2 were dose-dependent (EC50 of 293 ± 13 nM) and reversed by the
selective CB1 antagonist SR 141716A. In agreement with a presynaptic
localization of CB1 receptors, WIN-2 increased paired-pulse
facilitation, decreased miniature EPSC (mEPSC) frequency, and
had no effect on the mEPSCs amplitude. Perfusion with the adenylate
cyclase activator forskolin enhanced glutamatergic transmission but did
not alter presynaptic CB1 actions, suggesting that cannabinoids inhibit
glutamate release independently from the cAMP-PKA cascade. CB1 did not
reduce evoked transmitter release by inhibiting presynaptic
voltage-dependent Ca2+ currents through N-, L-, or
P/Q-type Ca2+ channels, because CB1 inhibition
persisted in the presence of  -Conotoxin-GVIA, nimodipine, or
-Agatoxin-IVA. The K+ channel blockers
4-aminopyridine (100 µM) and BaCl2 (300 µM) each reduced by 40-50% the inhibitory actions of
WIN-2, and their effects were additive. These data suggest that CB1
receptors are located on the cortical afferents to the nucleus and can
reduce glutamate synaptic transmission within the NAc by modulating
K+ channels activity.
Key words:
nucleus accumbens; cannabinoid; glutamate; CB1 receptors; presynaptic inhibition; K+ channels; mice
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INTRODUCTION |
Derivatives of Cannabis
sativa (L.), such as marijuana and hashish, have been used for
centuries for therapeutic and recreational purposes. The
psychopharmacologically active component of C. sativa, ( )-trans-delta9-tetrahydrocannabinol, as well as
cannabimimetics and endocannabinoids, mediate their actions in the
CNS through specific interactions with a
Gi/Go-protein-coupled
receptor [cannabinoid receptor (CB1)] (Mechoulam et al.,
1996 ). The CB1 receptor is widely expressed in the brain and has been
shown to inhibit adenylate cyclase (AC), activate
mitogen-activated protein kinases, reduce Ca2+ currents, and modulate several
K+ conductances (Mackie and Hille, 1992;
Mackie et al., 1993, 1995; Bouaboula et al., 1995; Twitchell et al.,
1997; Schweitzer, 2000). Activation of CB1 receptors inhibits synaptic
transmission in the hippocampus (Sullivan, 1999; Hoffman and Lupica,
2000), substantia nigra pars compacta (Chan and Yung, 1998), the
cerebellum (Levenes et al., 1998), and the prefrontal cortex (Auclair
et al., 2000).
The mesolimbic-mesocortical dopaminergic system and particularly the
nucleus accumbens (NAc) are essential to the reinforcing properties of
addictive drugs (Hyman, 1996; Koob, 1996). Drugs of abuse, such as
psychostimulants, opiates, nicotine, alcohol, and cannabinoids, alter
dopamine levels in the NAc (Kalivas and Duffy, 1990; Self and Nestler,
1995; Tanda et al., 1997; Pontieri et al., 1998). Because of intrinsic
and network properties, the projection cells of the NAc, the GABAergic
medium-spiny neurons, depend on glutamatergic excitatory afferents to
generate action potentials (Pennartz et al., 1994). Glutamatergic
transmission in the NAc participates in the effects of opiates and
psychostimulants (Pulvirenti et al., 1989, 1991, 1992; Pap and
Bradberry, 1995; Cornish and Kalivas, 2000) and is altered by chronic
drug treatment (Nie et al., 1994; Pierce et al., 1996a,b; Manzoni et
al., 1998). Although cannabis derivatives are the most common illicit
drugs, little is known of their actions in the mesolimbic regions and the NAc. In particular, the potential effects of cannabinoids on the
excitatory afferents to the NAc have never been explored.
The specific purpose of this study was to identify the localization and
functions of CB1 receptors at the glutamatergic cortical-NAc synapses.
Using immunohistochemical techniques, we identified CB1 receptors on
afferents making synaptic-like contacts with GABAergic neurons
(presumably medium spiny neurons) of the NAc. It was found that CB1
receptors inhibit glutamatergic excitatory synaptic transmission
through the modulation of presynaptic K+ conductances.
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MATERIALS AND METHODS |
Immunohistochemistry. After deep anesthesia with
sodium pentobarbital (50 mg/kg), five C57BL/6 male mice were perfused
through the ascending aorta with PBS, pH 7.4, followed by 300 ml
of fixative composed of 4% paraformaldehyde and 0.5% glutaraldehyde
in 0.1 M phosphate buffer, pH 7.4. The brain was
then dissected and fixed by immersion in the fixative without
glutaraldehyde for 12 hr at 4°C. It was then cut sagittally with a
vibratome into 40- to 50-µm-thick sections. These were carefully
rinsed in PBS and treated for single- or double-fluorescence
immunostaining. Sections were incubated for 48 hr at 4°C with either
one or two specific antibodies including (1) a rabbit polyclonal
antibody against CB1 cannabinoid receptor (diluted 1:500; kindly
provided by Dr. K. Mackie, Department of Anesthesiology, University of
Washington, Seattle, WA), or (2) both the anti-CB1 and a mouse
monoclonal antibody against GABA (diluted 1:1000; Chemicon, Temecula,
CA). After careful rinsing in PBS, sections were incubated for 2 hr with an anti-rabbit IgG conjugated with Cy3 (diluted 1:1000; The
Jackson Laboratory, Bar Harbor, ME), alone (single
immunostaining), or in combination with an anti-mouse IgG conjugated
with fluorescein isothiocyanate (FITC) (diluted 1:200; Sigma, St.
Louis, MO). After rinsing, the sections were mounted in mowiol
(Calbiochem, La Jolla, CA) and examined under a Bio-Rad (Hercules, CA)
MCR 1024 confocal laser scanning microscope equipped with a
krypton-argon mixed gas laser. Two laser lines emitting at 488 and 568 nm were used for exciting the FITC- and Cy3-conjugated secondary
antibodies, respectively. The organization of immunostained (IS)
structures was studied either 1/ on single confocal images 1- to
2-µm thick, or 2/ on reconstructed sections made by projecting
z-series of 20-40 consecutive confocal images 1 µm apart, collected
throughout the thickness of the vibratome section. The background noise
of each confocal image was reduced by averaging five image inputs.
Unaltered digitized images were transferred to a personal
computer, and PowerPoint (Microsoft, Seattle, WA) was used to prepare
and print final figures. The specificity of the antibodies has been
assessed previously by the absorption test (see Tsou et al., 1998 for
anti-CB1 and Szabat et al., 1992 for anti-GABA). Additional controls
consisted of omitting the primary antibodies and applying the secondary antibodies alone.
Electrophysiology. Whole-cell patch-clamp and extracellular
field recordings were made from medium spiny neurons in parasagittal slices of mouse NAc. This method has been described previously (Manzoni
et al., 1998). In brief, mouse (male C57BL/6, 4-6 weeks) were
anesthetized with fluorene and decapitated. The brain was sliced
(300-400 µm) in the parasagittal plane using a vibratome and
maintained in physiological saline at 4°C. Slices containing the NAc
were stored at least 1 hr at room temperature before being placed in
the recording chamber and superfused (2 ml/min) with artificial
CSF (ACSF) that contained (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl2, 2.4 CaCl2, 18 NaHCO3, 1.2 NaH2PO4, and 11 glucose, equilibrated with 95% O2-5%
CO2. All experiments were done at room
temperature. The superfusion medium contained picrotoxin (100 µM) to block GABAA
receptors. All drugs were added at the final concentration to the
superfusion medium.
For field potential recordings, the recording pipette was filled with a
3 M NaCl solution, and both the field EPSP (fEPSP) slope
(calculated with a least square method) and fEPSP amplitude were measured.
For patch-clamp experiments, cells were visualized using an
upright microscope with infrared illumination, and recordings were made
with whole-cell electrodes containing the following (in
mM): 128 Cs-gluconate, 20 NaCl, 1 MgCl2, 1 EGTA, 0.3 CaCl2, 2 Mg-ATP, 0.3 GTP, and 0.2 cAMP buffered with 10 HEPES, pH 7.3. Electrode
resistance was 4 M , acceptable access resistance was <15 M, and
the holding potential was 70 mV. An Axopatch-1D (Axon Instruments,
Foster City, CA) was used to record the data, which were filtered at
1-2 kHz, digitized at 5 kHz on a DigiData 1200 interface (Axon
Instruments), and collected on a personal computer using ACQUIS-1
software (Bio-Logic, St. Egreve, France). To evoke synaptic
currents, stimuli (100-150 µsec duration) were delivered at 0.033 Hz
through bipolar tungsten electrodes placed at the prefrontal
cortex-accumbens border (Manzoni et al., 1997, 1998). Recordings were
made in the rostromedial dorsal accumbens close to the anterior
commissure. Evoked EPSC amplitudes were measured by averaging a 5 msec
window around the peak and subtracting the average value obtained
during a 10 msec window immediately before the stimulus. Two stimuli
were applied at an interval of 50 msec, and the paired-pulse ratio
(PPR) was calculated by dividing the amplitude of the EPSC evoked by
the second stimulus by the amplitude of the first EPSC evoked by the
first stimulus. A change in the paired-pulse ratio is thought to result
from the alteration in transmitter release caused by a presynaptic
mechanism (Manabe et al., 1993).
Miniature EPSCs (mEPSCs) were recorded in the presence of tetrodotoxin
(TTX) (300 nM) using Axoscope 1.1.1. mEPSC amplitude and
inter-interval time were measured using Axograph 3.6. For this
analysis, a template of mEPSCs having the width and time course of a
typical synaptic event [a double exponential:
f(t) = exp(t/Rise) exp(t/Decay), where rise is 0.5 msec, and decay is 3 msec] was slid along the data trace one point at a time. At each
position, this template is optimally scaled and offset to fit the data,
and a detection criterion is calculated. The detection criterion is the
template scaling factor divided by the goodness-of-fit at each
position. An event is detected when this criterion exceeds a threshold
and reaches a sharp maximum. The limit of detection was 2 pA (Manzoni
and Williams, 1999).
The fitting of concentration-response curves were calculated according
to y = {ymax ymin/1 + (x/EC50)n} + ymin (where
ymax is response in the absence of
agonist, ymin is response remaining in
the presence of maximal agonist concentration, x is
concentration, EC50 is concentration of agonist
producing 50% of the maximal response, and n is the slope)
with Kaleidagraph software (Abelbeck Software, Readinig, PA). All
values are given as mean ± SEM. Statistical analyses were done
with the Mann-Whitney U test or the Kolmogorov-Smirnov tests using Statview (Abacus Concepts, Calabasas, CA), and
p < 0.05 was taken as indicating statistical
significance. The drugs used were as follows: WIN 55,212,2 (WIN-2), WIN
55,212,3, and CP 55940 were from Tocris Cookson (Bristol, UK); TTX,
picrotoxin, BaCl2, CdCl2,
forskolin, adenosine, 4-AP, and nimodipine were from Sigma (St. Quentin
Fallavier, France); -Agatoxin-IVA and -Conotoxin GVIA were from
Alomone Labs (Jerusalem, Israel); and SR 141716A was generously
provided by Sanofi Recherche (Montpellier, France).
WIN-2, WIN 55,212,3, CP 55940, and SR 141716A were prepared
as (10 mM) stock solutions in DMSO. Final concentrations
were <0.1% DMSO.
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RESULTS |
Throughout the brain, the organization of the CB1-IS structures
conformed to previous descriptions in the rat (Tsou et al., 1998).
Dispersed CB1-IS perikarya were observed throughout the cortex, the
hippocampus, and the olfactory bulb. More specifically, numerous CB1-IS
perikarya were detected in the prelimbic cortex area (Fig.
1A), which knowingly
massively projects to the NAc (Wright and Groenewegen, 1995). Dense
plexuses of intensely labeled CB1-IS fibers were detected throughout
the cortex, the hippocampus, the anterior olfactory nucleus, and the
olfactory bulb. Although more dispersed than in these regions, a number
of CB1-IS fibers were also detected throughout the striatum. Within the
NAc, these CB1-IS fibers were mostly localized to the core of the
nucleus in which they appeared as large, poorly branched fibers
exhibiting a large number of intensely immunostained varicosities (Fig.
1B). Double-immunostaining experiments indicated
that, throughout the NAc, these CB1-IS fibers established frequent en
passant or terminal synaptic-like contacts with GABA-IS perikarya or
dendritic processes (Fig. 1C,D).
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Figure 1.
Localization of CB1 receptors in the prelimbic
cortex and the nucleus accumbens. Confocal images of single- or
double-immunostained sections. A, Single immunostaining
for CB1 shows that intense labeling is associated with perikarya
located in the prelimbic cortex. B, CB1 labeling was
also associated with large varicose axonal fibers that extend
throughout the core of the NAc surrounding the anterior commissure.
C, D, Double immunostaining for both CB1
(red) and GABA (green) shows that,
within the NAc, CB1-IS fibers form terminal (arrows in
C) or en passant (arrows in
D) synaptic-like contacts with GABA-IS perikarya.
AC, Anterior commissure. Scale bars, 25 µm.
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The present immunohistochemical identification of presynaptic CB1
receptors on axonal fibers in the NAc prompted us to explore the
effects of cannabimimetics at the glutamatergic synapses between the
prelimbic cortex and the NAc.
Extracellular field potential recordings were performed to measure the
effects of CB1 agonists on synaptic responses evoked by stimulating
prelimbic cortex fibers (Manzoni et al., 1997, 1998). It was found that
fEPSPs, in the core of the NAc, were strongly inhibited by CB1
agonists. Bath-applied WIN-2 (10 µM) reduced the fEPSP to
35.3 ± 5.8% of its basal value (n = 12) (Fig. 2A). This depression
was strongly reversed by the selective CB1 antagonist SR 141716A (10 µM) (Rinaldi-Carmona et al., 1994), suggesting
the implication of cannabinoid receptors of the CB1 subtype (Fig.
2A). We also verified that the WIN-2-induced
inhibition was totally prevented by preincubation with the CB1
antagonist SR 141716A (Fig. 2B).
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Figure 2.
CB1 receptor-mediated inhibition of evoked
excitatory synaptic transmission in mice nucleus accumbens.
A, The cannabimimetic WIN-2 (10 µM)
reduced the fEPSP on average to 34 ± 5% (n = 12) of its basal value. Traces represent averages of 10 consecutive EPSPs. The effects of WIN-2 were reversed by the selective
CB1 antagonist SR 141716A (10 µM). B,
Preincubation of the slices with 10 µM of the CB1
antagonist SR 141716A was without effect on basal synaptic transmission
(data not shown) but completely prevented the WIN-2-induced inhibition
(n = 5).
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To further confirm the implication of CB1 receptors, the following
experiments were performed. First, we determined that the effects of
WIN-2 were dose-dependent with an EC50 of
291 ± 13 nM (Fig.
3A). Second, because high
doses (1-10 µM) of WIN-2 were routinely used
to ensure fast and complete drug penetration into the slices, we
studied the effects of WIN 55,212,3, the enantiomer of WIN-2, which
lacks affinity for cannabinoid binding sites. Figure 3B
shows that, contrary to the WIN-2-induced inhibition, the effects of
WIN 55,212,3 were not blocked by a selective CB1 antagonist. Finally,
the WIN-2 effect on fEPSP was not modified by preincubation with
saturating doses of WIN 55,212,3 (Fig. 3B). These
experiments confirmed the specificity of WIN-2 for CB1 receptors in the
NAc slice preparation. Third, it was verified that the CB1 agonist CP
55940 (2 µM), which does not relate
structurally to WIN-2, also reduced the fEPSP (Fig. 3C).
Together, these data strongly suggest that the inhibitory effects of
WIN-2 can be attributed to the activation of CB1 receptors.
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Figure 3.
Pharmacological characterization of the
CB1-mediated inhibition. A, Dose-response curve for the
CB1 agonist WIN-2. The EC50 for WIN-2 was 291 ± 13 nM. Each point is expressed as the
percentage of inhibition of basal evoked transmission, and the error
bar represents the SEM. B, WIN 55,212,3 (10 µM), an enantiomer of WIN-2 inactive at CB1 binding
sites, caused a small but significant fEPSP inhibition that, contrary
to the WIN-2-induced inhibition, was not reversed by SR 141716A (10 µM; black bar). WIN-2-mediated fEPSP
reduction was additive to the WIN 55,212,3 effect (hatched
bar). These data are in agreement with the idea that WIN-2 at
concentrations up to 10 µM is selective for CB1
receptors. C, The cannabinoid agonist CP 55940 (2 µM; n = 5), which is not structurally
related to WIN-2, also inhibited fEPSP.
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Our immunohistochemical data demonstrated the association of CB1
immunostaining with axonal fibers innervating the NAc and naturally
suggest a presynaptic site of action. In a first attempt to
functionally assess the origin of the CB1-mediated depression, the
variation of the PPR of excitatory transmission, a presynaptic phenomenon thought to reflect changes in transmitter release and sensitive to presynaptic manipulations, was measured. Bath application of 10 µM WIN-2 induced a depression of EPSCs recorded in
the whole-cell patch-clamp configuration (Cs-gluconate-based
intracellular medium, holding potential of 70 mV). The depression of
evoked release was accompanied by an increase of the PPR. At the peak
of the depression induced by WIN-2, the EPSC was reduced to 51 ± 11% of its control value, whereas the PPR was up to 169 ± 46%
of its control value (n = 6). This finding suggested
that CB1 receptors could decrease presynaptic neurotransmitter release.
To further characterize the site of action of CB1 receptors, mEPSCs
were recorded. A decrease in the frequency of the mEPSCs is interpreted
to be a result of a presynaptic action (e.g., a reduction in
transmitter release), whereas a decrease in mEPSCs amplitude
classically reveals a postsynaptic site of action (Katz, 1966). We
first verified that a high concentration of the
Ca2+ channel blocker cadmium (100 µM) did not affect mEPSCs frequency or amplitude recorded
in the presence of TTX (Fig.
4A,B). This showed that, in the NAc, mEPSCs are totally independent of external Ca2+ entry. What are the effects of WIN-2
on the Ca2+-independent mEPSCs? A typical
experiment shows that, in the presence of TTX, the mEPSCs frequency was
depressed by WIN-2, whereas the mEPSCs amplitude remained unaffected
(Fig. 4C). Accordingly, the distribution of the
mEPSCs amplitude was not modified by WIN-2 (Fig. 4D),
whereas the time interval distribution was shifted to the right (Fig.
4E). These data suggest that CB1-mediated inhibition of action potential-independent synaptic transmission does not require
interactions with presynaptic Ca2+
channels. In all cases, the experiments demonstrated that the CB1-mediated inhibition is mediated by presynaptic receptors.
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Figure 4.
CB1-induced inhibition of action
potential-independent glutamate release reveals a presynaptic site of
action. A, B, Miniature EPSCs recorded in
the presence of TTX are independent of Ca2+ channels
activation. Bath application of cadmium chloride (100 µM)
to block voltage-sensitive Ca2+ channels changed
neither the amplitude distribution (A) nor the
frequency distribution of mEPSCs (B).
C, Representative consecutive 1 sec current sweeps from
a cell (holding potential of 70 mV) in which mEPSCs were recorded in
the absence or presence of 10 µM WIN-2. D,
The distribution of mEPSC amplitude before and during the application
of the CB1 agonist, in the seven cells recorded, was unchanged after 20 min bath perfusion of WIN-2. E, The distribution of the
time intervals between successive mEPSCs in all of the neurons recorded
(same as above) revealed that the mEPSCs frequency was reduced during
WIN-2 application. For control conditions, a total of 1849 events were
detected over a period of 9.14 ± 1.14 min (n = 7; range, 6-14 min). In the same neurons, a total of 1004 events
were collected after 10-15 min WIN-2 perfusion over a period of
11.86 ± 2.20 min (range, 6-20 min).
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What are the presynaptic targets of CB1 receptors? How do they inhibit
synaptic transmission at the cortical afferents to the NAc?
Theoretically, CB1 receptors could inhibit synaptic release of
glutamate because of their negative coupling to AC types I, III, V, VI,
and VIII (Rhee et al., 2000) and the resulting decrease in
intracellular cAMP levels, either through the inhibition of presynaptic
voltage-sensitive calcium channels (VSCCs), which would decrease
intraterminal Ca2+ levels and reduce
evoked synaptic transmission (Mackie and Hille, 1992; Chan and Yung,
1998; Sullivan, 1999; Hoffman and Lupica, 2000) or through the
activation of potassium channels that would cause a strong
hyperpolarization of the synaptic terminal ("synaptic shunt")
(Deadwyler et al., 1995; Mackie et al., 1995; McAllister et al.,
1999).
To determine whether the AC-cAMP pathway interacted with the
CB1-induced synaptic depression, the effects of forskolin, a powerful
activator of AC (Seamon and Daly, 1986), were examined. Bath
application of forskolin (10 µM) reliably enhanced NAc
fEPSPs of ~50% (Fig. 5A).
When the forskolin effect had reached its plateau, WIN-2 was perfused
and induced a depression identical to what was observed in control
conditions (Fig. 5B). Thus, in the NAc, CB1 receptors
inhibit glutamate synaptic release independently of cAMP levels.
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Figure 5.
CB1-mediated inhibition is independent of cAMP
levels. A, The adenylate cyclase activator forskolin
(FSK; 10 µM, 20 min) caused a robust
augmentation of the fEPSP in NAc slices (n = 9).
B, The WIN-2-mediated fEPSP inhibition was not affected
when cAMP levels were augmented by bath application of forskolin
(n = 7). Forskolin was applied 20 min before and
during the WIN-2 application (10 µM).
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The fact that CB1 activation could inhibit the
Ca2+-independent release of glutamate in
the NAc (Fig. 4) did not exclude an additional interaction with the
Ca2+ channels underlying evoked synaptic
release. In fact, in the hippocampus, CB1 receptors have been shown to
inhibit evoked glutamatergic and GABAergic synaptic transmission via an
interaction with VSCCs (Chan and Yung, 1998; Sullivan, 1999; Hoffman
and Lupica, 2000). It was therefore decided to alter presynaptic
Ca2+ entry by blocking L-, N-, or P/Q-type
Ca2+ channels with nimodipine,
-Conotoxin-GVIA, or -Agatoxin-IVA, respectively. Figure
6A shows in a
representative experiment that, similar to what we described previously
in the rat NAc (Manzoni et al., 1997), -Conotoxin-GVIA-sensitive
Ca2+ channels (presumably of the N-type)
are responsible for most of the evoked transmission in mice NAc. All of
the experiments performed with -Conotoxin-GVIA (1 µM, 15-20 min) gave similar results and are
summarized Figure 6B. In the presence of
-Conotoxin-GVIA (1 µM), WIN-2 depressed the
fEPSP to a similar extent as in control conditions. Similarly, bath
perfusion with 200 nM -Agatoxin-IVA or 1 µM nimodipine to selectively block the P/Q or
L-type channels, respectively, reduced by 15-20% synaptic
transmission but did not affect the inhibitory actions of WIN-2 (Fig.
6C). It was concluded that, in the NAc, the CB1-induced
depression does not require the reduction of presynaptic
Ca2+ entry through N-, P/Q-, or L-type
channels.
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Figure 6.
The WIN-2-induced inhibition does not require N-,
L-, or P/Q-type Ca2+ channels modulation.
A, Typical experiment in which the slice was perfused
with 1 µM -Conotoxin-GVIA, which blocked ~60% of
the fEPSP. It is clear that the fraction of synaptic transmission
insensitive to -Conotoxin-GVIA was still sensitive to the CB1
agonist WIN-2 (10 µM). B, Summary of all
of the experiments performed as above with specific blockers of N-type
(-conotoxin-GVIA, 1 µM), L-type (nimodipine, 1 µM); and P/Q-type (-Agatoxin-IVA, 200 nN)
voltage-sensitive Ca2+ channels. The histogram of
the maximum inhibitions caused by perfusion of selective agents reveals
how the different types of voltage-sensitive Ca2+
channels contribute to the evoked release of glutamate in the NAc.
C, Histogram of the maximum 10 µM
WIN-2-mediated inhibition in the presence of Ca2+
channel inhibitors. All experiments were performed as in
A.
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Voltage-dependent and -independent
K+ channels are modulated by CB1 receptors
(Deadwyler et al., 1995; Henry and Chavkin, 1995; Mackie et al., 1995;
Garcia et al., 1998; McAllister et al., 1999). Therefore, the actions
of K+-channels blockers on the
cannabimimetics-induced presynaptic inhibition were assessed. Bath
application of 4-AP (100 µM), BaCl2 (300 µM), or the combination of both caused a large
enhancement of the duration and the size of the evoked fEPSP. When
voltage-dependent K+ conductances were
blocked by 4-AP (100 µM), the WIN-2-induced inhibition
was significantly reduced (Fig.
7A). Blockade of
K+ conductances with
BaCl2 (300 µM) also
caused a clear reduction in the inhibitory actions of the
cannabimimetic (Fig. 7A), consistent with the involvement of
G-protein-gated inwardly rectifying K+
channel-like conductances (Coetzee et al., 1999). When the
K+ channels blockers were applied
together, the inhibitory actions of WIN-2 were completely prevented
(Fig. 7A). Notably, in the same experiments, adenosine (200 µM) could still inhibit fEPSPs, suggesting that
presynaptic inhibitory processes were still intact [43 ± 3 (n = 3) and 45 ± 6% (n = 5)
inhibition of the fEPSP in control and
4-AP-BaCl2, respectively] (Fig. 7A).
Nonetheless, an important limitation in the interpretation of these
experiments is that blockade of presynaptic
K+ channels augments presynaptic action
potential duration and could cause the saturation of the
Ca2+-dependent release process (Colmers et
al., 1988; Hoffman and Lupica, 2000). Thus, it is possible that the
lack of effect of WIN-2 in the presence of
K+ channels blockers is
attributable to indirect actions on some Ca2+-dependent processes. To address this
question, [Ca2+]o
was lowered (from 2.4 to 0.3-1.2 mM, with
corresponding increases in Mg2+
concentrations to correct for the osmolarity change) to reduce the size
of evoked EPSPs in the presence of 4-AP and
BaCl2. Lowering [Ca2+]o, although
efficient at reducing fEPSPs evoked in the presence of 4-AP and
BaCl2, did not restore WIN-2-induced inhibition
(Fig. 7B,C). We verified that, in
low [Ca2+]o, the
WIN-2 inhibition was identical to what was observed in standard
extracellular medium (Fig. 7C). Together, these data demonstrate that presynaptic CB1 receptors modulate synaptic
transmission through the modulation of K+
conductances.
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Figure 7.
Effects of K+ channels blockade
on the CB1-induced inhibition of glutamatergic synaptic transmission in
the NAc. A, In standard ACSF (2.4 mM CaCl2;
white bar), 4-AP (100 µM; hatched
bar), and BaCl2 (300 µM;
dotted bar) reduced the WIN-2-induced fEPSP inhibition.
WIN-2 reduced fEPSP by 39.1 ± 5.5% (n = 5;
p = 0.015) and 32.3 ± 9.4%
(n = 4; p = 0.029) in the
presence of 4-AP and BaCl2, respectively, compared
with 65.6 ± 5.4% (n = 12) in control. When
added together, 4-AP and BaCl2 (black bar)
completely prevented the WIN-2 (10 µM) inhibition
(1.7 ± 11%; n = 4; p = 0.002). In contrast, the adenosine (200 µM; white
bar)-induced inhibition was not affected by pretreatment with
4-AP and BaCl2 (black
bar). B, Representative experiment in which the
large enhancement of the fEPSP caused by the combination of 4-AP and
BaCl2 was reduced by lowering extracellular
Ca2+ concentration from 2.4 to 0.3 mM.
In this condition, 4-AP and BaCl2 still prevented the 1 µM WIN-2-induced inhibition. C, Summary of
all the experiments performed as above (n = 8). It
is clear that 4-AP (100 µM) and BaCl2 (300 µM) blocked the CB1-mediated inhibition in low external
Ca2+. Lowering the external Ca2+
affected neither the time course nor the amplitude of the WIN-2 (1 µM)-mediated inhibition of the fEPSP [compare the
inhibition in control ACSF (filled circles) with
the inhibition in low external Ca2+ (open
circles)].
|
|
| |
DISCUSSION |
The results show that, in the mouse NAc, CB1 receptors are present
on large fibers making synaptic-like contacts with GABA containing
perikarya or processes. It was found that cannabimimetics, with a
pharmacology consistent with the involvement of CB1 receptors, caused a
profound inhibition of glutamatergic transmission at the synapses
between the prelimbic cortex and the NAc. The electrophysiological analysis corroborated the anatomical data and pointed out a presynaptic site of action for CB1 receptors. It is also shown that the CB1 receptor-mediated presynaptic inhibition of glutamatergic transmission is independent of the cAMP-PKA cascade and of the inhibition of VSCCs.
These data thereby are in striking contrast to those showing that the
modulation of presynaptic VSCCs is responsible for the CB1
receptor-mediated inhibition of transmitter release in the hippocampus
(Sullivan, 1999; Hoffman and Lupica, 2000) and substantia nigra pars
compacta (Chan and Yung, 1998). Finally, we provide evidence for a role
of voltage-sensitive and -insensitive K+
channels in mediating CB1 presynaptic inhibition of glutamate release
because low concentrations of the potassium channel blockers 4-AP and
BaCl2 completely prevented the CB1 effect.
The effects of CB1 receptors are presynaptic
CB1 receptors have been found in axons, cell bodies,
and dendrites (Herkenham et al., 1991; Tsou et al., 1998) and could
theoretically modulate synaptic transmission at presynaptic and
postsynaptic sites via a variety of cellular effectors. Practically,
when CB1-mediated inhibition of synaptic transmission was observed in
the CNS, it always resulted from presynaptic actions
(Levenes et al., 1998; Shen and Thayer, 1998; Sullivan, 1999; Auclair
et al., 2000; Hoffman and Lupica, 2000). Before this study, no data
were available on the presence of CB1 receptors at excitatory or
inhibitory synapses of the NAc. We present compelling evidences for a
presynaptic site of action of CB1 receptors at the excitatory synapses
to the NAc medium spiny neurons. First, we identified immunostaining for CB1 receptors on axons making synaptic-like contacts to GABAergic neurons of the NAc (Fig. 1C,D). Intense CB1
immunostaining was located within the cytoplasm of a number of
perikarya dispersed throughout the prelimbic cortex (Fig.
1A). Although suggestive of a cortical localization,
our observation does not exclude other origins for the CB1 receptors
containing neurons making synapses in the NAc (e.g., hippocampus,
amygdala... ). Second, electrophysiological analysis showed an
increase in the paired-pulse ratio of evoked EPSCs and a decrease of
the mEPSC frequency (but not their amplitude) during CB1 inhibition
(Fig. 4). Thus, the simplest interpretation of our anatomical and
electrophysiological data are that a presynaptic mechanism is
responsible for the actions of the cannabinoid agonists at the
excitatory synapses to the NAc.
As shown previously for GABAB and adenosine A1
receptors (Scanziani et al., 1992; Wu and Saggau, 1994; Dittman and
Regehr, 1996), the CB1 receptor seems to activate two separate and
inhibitory mechanisms in the terminal; one is likely to involve a
direct action on the release machinery and is responsible for the
reduction of action potential independent transmitter release, whereas
the other one would involve one or more K+
conductances and participates to the inhibition of evoked transmitter release (see below).
The CB1 effect is independent of the cAMP-PKA cascade
Like many other receptors coupled to pertussis
toxin-sensitive G-protein, CB1 receptor actions include inhibition of
adenylyl cyclase. Some of the effects of cannabinoid withdrawal depend on the activation of the cAMP-PKA cascade (Tzavara et al., 2000), and
chronic treatment with CB1 agonists causes a superactivation of several
adenylyl cyclases isozymes (Rhee et al., 2000), underlying the
importance of the cAMP-PKA cascade in the long-term effects of
cannabinoids. The acute inhibition of adenylyl cyclase by CB1 receptors
can have several synaptic consequences, mainly through the modulation
of presynaptic ion channels or the direct inhibition of transmitter
release. In the guinea pig myenteric plexus, elevation of cAMP levels
by the adenylyl cyclase activator forskolin significantly reduced the
maximum inhibitory response to WIN-2 (Coutts and Pertwee, 1998). In
agreement with the idea that presynaptic inhibition mediated through
the adenylyl cyclase cascade are not universal at CB1 sensitive
synapses, the CB1 inhibition was not occluded in forskolin-treated
slices (Fig. 5B).
The CB1-mediated inhibition of synaptic transmission does
not require voltage-sensitive Ca2+ channels
One of the most documented actions of CB1 receptors in neuronal
cells is their inhibitory coupling with voltage-sensitive Ca2+ channels (Caulfield and Brown, 1992;
Mackie and Hille, 1992; Mackie et al., 1993, 1995; Pan et al., 1996;
Twitchell et al., 1997; Shen and Thayer, 1998). Not surprisingly,
recordings from substantia nigra pars reticulata neurons showed that
cannabinoids exert a presynaptic inhibition on GABAergic transmission
via the CB1 inhibition of Cd2+-sensitive
presynaptic Ca2+ channels (Chan and Yung,
1998). More recently, it was shown that Cd2+ prevented cannabinoids actions
on spontaneous GABA release (Hoffman and Lupica, 2000) and that CB1
receptors can inhibit evoked glutamate release via the modulation of N-
and P/Q-type Ca2+ channels (Sullivan,
1999). In marked contrast, we found that CB1 receptors inhibit action
potential-independent and evoked synaptic transmission independently
from the modulation of presynaptic voltage-sensitive
Ca2+ channels (Fig. 6).
A role for K+ channels in mediating the CB1
induced inhibition of glutamate release
Modulation of voltage-dependent and -independent potassium
conductances is another well described effect of CB1 receptors (Deadwyler et al., 1995; Henry and Chavkin, 1995; Mackie et al., 1995;
Garcia et al., 1998; McAllister et al., 1999; Schweitzer, 2000).
Moreover, recent reports have underlined the importance of
K+ conductances in mediating some opioid
receptor actions on inhibitory (Vaughan et al., 1997) and excitatory
synaptic transmission (Simmons and Chavkin, 1996; Manzoni and Williams,
1999). Surprisingly, it was found that blockade of presynaptic
K+ channels hampered the cannabinoid
effects in standard conditions but also when
[Ca2+]o was
lowered to reduce the size of evoked EPSPs and control for indirect
actions of K+ channel blockers on
Ca2+-dependent processes (Fig. 7). Similar
experiments performed at the GABAergic synapses of the CA1 region of
the hippocampus led to opposite results because lowering
[Ca2+]o restored
the inhibitory properties of WIN-2 (Colmers et al., 1988; Hoffman and
Lupica, 2000). It is likely that this apparent discrepancy is simply an
example of the diversity of the cellular effectors of CB1 receptors at
the synapses of the CNS.
Conclusions
How do cannabinoids activate mesolimbic dopamine
neurons (Gessa et al., 1998) and increase dopamine levels in the NAc
(Tanda et al., 1997; Szabo et al., 1999)? In substantia nigra pars
reticulata, the presence of CB1-immunoreactive fibers (Tsou et al.,
1998) suggests that disinhibition of GABAergic afferents participates to the net excitatory actions of cannabinoids. In contrast, CB1 receptors are absent from the ventral tegmental area (VTA) (Herkenham et al., 1991; Tsou et al., 1998) and the present demonstration of CB1
receptors at the glutamatergic synapses in the NAc provides another
means for cannabinoids to excite dopaminergic neurons. The
glutamatergic afferents to the NAc control the firing of the NAc
GABAergic neurons, which in turn inhibit the dopaminergic neurons of
the VTA. Via the reduction of excitatory transmission in the NAc,
cannabinoids could disinhibit dopamine cells of the VTA, increase their
firing rate, and trigger the release of dopamine in the nucleus accumbens.
| |
FOOTNOTES |
Received Aug. 11, 2000; revised Oct. 11, 2000; accepted Oct. 12, 2000.
This research was supported by Centre National de la Recherche
Scientifique, Institut National de la Santé et de la Recherche Médicale, Economic European Community BIOTECH and BIOMED,
and the Bayer Company. Work in the laboratory of O.J.M. was supported by grants from Mission Interministérielle à la Lutte contre la Drogue et la Toxicomanie and by Ministère de la Recherche (Action Concertée Incitative "Jeunes Chercheurs"). We thank
Dr. K. Mackie for his generous gift of the anti-CB1 antibodies and M. Passama for the artwork.
Correspondence should be addressed to Dr. O. Manzoni, Centre National
de la Recherche Scientifique, Unité Propre de Recherche 9023, 141, Rue de la Cardonille, 34094 Montpellier Cedex 05, France. E-mail:
manzoni{at}ccipe.montp.inserm.fr.
| |
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TOP
ABSTRACT
INTRODUCTION
