Cellular Neurobiology Branch, National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health, Baltimore, Maryland 21224
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ABSTRACT |
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Hoffman, Alexander F. and
Carl R. Lupica.
Direct Actions of Cannabinoids on Synaptic Transmission in
the Nucleus Accumbens: A Comparison With Opioids.
J. Neurophysiol. 85: 72-83, 2001.
The nucleus accumbens
(NAc) represents a critical site for the rewarding and addictive
properties of several classes of abused drugs. The medium spiny
GABAergic projection neurons (MSNs) in the NAc receive innervation from
intrinsic GABAergic interneurons and glutamatergic innervation from
extrinsic sources. Both GABA and glutamate release onto MSNs are
inhibited by drugs of abuse, suggesting that this action may contribute
to their rewarding properties. To investigate the actions of
cannabinoids in the NAc, we performed whole cell recordings from MSNs
located in the shell region in rat brain slices. The cannabinoid
agonist WIN 55,212-2 (1 µM) had no effect on the resting membrane
potential, input resistance, or whole cell conductance, suggesting no
direct postsynaptic effects. Evoked glutamatergic excitatory
postsynaptic currents (EPSCs) were inhibited to a much greater extent
by [Tyr-D-Ala2,
N-CH3-Phe4, Gly-ol-enkephalin] (DAMGO,
~35%) than by WIN 55,212-2 (<20%), and an analysis of miniature
EPSCs suggested that the effects of DAMGO were presynaptic, whereas
those of WIN 55,212-2 were postsynaptic. However, electrically evoked
GABAergic inhibitory postsynaptic currents (evIPSCs), were reduced by
WIN 55,212-2 in every neuron tested (EC50 = 123 nM;
60% maximal inhibition), and the inhibition of IPSCs by WIN 55,212-2 was completely antagonized by the CB1 receptor antagonist SR141716A (1 µM). In contrast evIPSCs were inhibited in ~50% of MSNs by the
µ/ opioid agonist
D-Ala2-methionine2-enkephalinamide
and were completely unaffected by a selective µ-opioid receptor
agonist (DAMGO). WIN 55,212-2 also increased paired-pulse facilitation
of the evIPSCs and did not alter the amplitudes of
tetrodotoxin-resistant miniature IPSCs, suggesting a presynaptic
action. Taken together, these data suggest that cannabinoids and
opioids differentially modulate inhibitory and excitatory synaptic
transmission in the NAc and that the abuse liability of marijuana may
be related to the direct actions of cannabinoids in this structure.
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INTRODUCTION |
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The nucleus accumbens/ventral
striatum (NAc) represents a critical site for mediating the rewarding
and/or addictive properties of several classes of abused drugs,
including ethanol, opioids, psychomotor stimulants, and marijuana
(Gardner and Vorel 1998; Koob 1992
;
Koob et al. 1998
; Wise 1996
; Wise
and Bozarth 1987
). It is generally appreciated that all of
these drugs augment extracellular dopamine levels in the NAc and that
this action contributes to their rewarding properties (Di Chiara
and Imperato 1988
; Koob 1992
; Koob et al.
1998
; Wise and Bozarth 1987
). However, recent evidence also suggests that many drugs of abuse have
dopamine-independent interactions with NAc neuronal circuitry
(Carlezon and Wise 1996
; Chieng and Williams
1998
; Koob 1992
; Martin et al.
1997
; Yuan et al. 1992
).
The majority (>90%) of neurons within the NAc are GABAergic medium
spiny neurons (MSNs) (Groenewegen et al. 1991) that send their output to several brain structures, including the ventral pallidum (Chang and Kitai 1985
) and the ventral
tegmental area (VTA) (Steffensen et al. 1998
). These
cells receive dopaminergic input from the VTA and glutamatergic inputs
from the hippocampus, amygdala, and prefrontal cortex (Christie
et al. 1985
, 1987
; Pennartz and Kitai 1991
).
Previous work has suggested that the synaptic inhibition of striatal
MSNs occurs via axon collaterals of the MSNs themselves (Park et
al. 1980
; Wilson and Groves 1980
). However, more
recent studies provide strong evidence that GABAergic inhibition is
mediated by intrinsic interneurons that comprise only a small fraction
of the striatal/NAc neuronal population (i.e., 3-5%) but provide
extensive innervation of medium spiny output neurons (Jaeger et
al. 1994
; Kawaguchi et al. 1995
;
Koós and Tepper 1999
).
Many commonly abused drugs, including opioids, psychomotor stimulants
and phencyclidine (PCP) are self-administered into the NAc by animals
(Carlezon and Wise 1996; McBride et al.
1999
) and inhibit fast amino acid-mediated synaptic
transmission in this structure (Chieng and Williams
1998
; Harvey and Lacey 1996
, 1997
; Martin
et al. 1997
; Nicola and Malenka 1997
). These two
observations suggest that at least part of the rewarding effects of
these drugs may be due to their direct effects on synaptic transmission
in the NAc. In addition, since dopamine itself inhibits both GABAergic and glutamatergic synaptic inputs to NAc MSNs (Harvey and Lacey 1996
, 1997
; Nicola and Malenka 1997
;
Pennartz et al. 1992
), it is possible that the direct
effects of these drugs on NAc circuitry, and their indirect effects via
an increase in NAc dopamine levels may contribute to the rewarding
properties of these drugs. Based on these data, we hypothesize that to
the extent that the inhibition of synaptic transmission in the NAc
reflects the rewarding properties of drugs of abuse, other drugs that
similarly modulate synaptic transmission in the NAc will also exhibit
rewarding properties.
Cannabis sativa (marijuana) is a drug possessing
pharmacological properties that sustain its use in humans (Abood
and Martin 1992). Although marijuana has been used for
centuries, it is only recently that its actions on neuronal circuitry
have begun to be understood. Its active constituents, termed
cannabinoids, activate various cellular effectors via interaction with
G-protein-coupled receptors classified as CB1 and CB2 (Ameri
1999
; Howlett 1995
; Pertwee
1997
). Whereas CB2 receptor distribution is restricted to
peripheral sites, the CB1 receptor is found extensively throughout the
mammalian CNS (Pertwee 1997
). Cannabinoids have been
shown to inhibit the release of several neurotransmitters, including glutamate (Misner and Sullivan 1999
; Shen et al.
1996
; Takahashi and Linden 2000
) and GABA
(Chan et al. 1998
; Hoffman and Lupica 2000
; Vaughan et al. 1999
, 2000
) in a variety of
brain areas. In addition to these actions, cannabinoids increase the
activity of midbrain dopamine neurons that project to the NAc
(French 1997
), increasing dopamine levels in this
structure (Chen et al. 1990
). Although the NAc contains
a moderately high-density of CB1 receptors (Herkenham et al.
1991
; Tsou et al. 1998
), the actions of these drugs in this structure remain largely unknown. Therefore in the present study we have examined the consequences of cannabinoid receptor
activation on the physiology of MSNs in the shell region of the NAc and
have compared these effects to those generated by opioid receptor activation.
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METHODS |
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All protocols were conducted under National Institutes of Health Guidelines using the handbook "Animals in Research" and were approved by the Institutional Animal Care and Use Committee (National Institute on Drug Abuse, Intramural Research Program, Baltimore, MD).
Slice preparation
Male Sprague-Dawley rats (Charles River Labs, Raleigh, NC),
14-30 days old, were killed by decapitation, and their brains were
rapidly removed and placed in ice-cold oxygenated artificial cerebral
spinal fluid (ACSF; see following text). The brain was then blocked in
a coronal plane ~3 mm anterior to, and 5 mm posterior to Bregma using
a razor blade. The posterior end of the tissue block was then glued to
the stage of a vibrating tissue slicer (Technical Products
International, St. Louis, MO) using cyanoacrylate. A midsaggital cut
was then made with a scalpel blade to separate the two hemispheres, and
coronal brain slices were cut at 300-µm nominal thickness. The slices
were then transferred to a beaker containing ACSF, aerated with 95%
O2-5% CO2 at room
temperature, where they were stored for 90 min before recordings.
Slices were transferred to a recording chamber (~250 µl volume)
that was integrated into the stage of an upright microscope (Carl Zeiss
Instruments, Germany). The slices were held in place by a circular
platinum-iridium wire and were continuously superfused with oxygenated
artificial cerebrospinal fluid (ACSF) at a rate of 2 ml/min, at room
temperature (~23°C). Control ACSF consisted of (in mM) 126 NaCl,
3.0 KCl, 1.5 MgCl2, 2.4 CaCl2, 1.2 NaH2PO4, 11.0 glucose, and
26 NaHCO3 and was saturated with 95%
O2 and 5% CO2.
Localization of MSNs
All MSNs included in this study were located within the shell
region of the NAc, contained in slices taken from ~1.6 to 0.7 mm
anterior to Bregma (Paxinos and Watson 1986). The
anterior commissure and the islands of Calleja were used as landmarks
for locating the shell region of the NAc. Thus recordings were made from MSNs found ~300-500 µm medial to the anterior commissure and
100-1200 µm dorsal to the islands of Calleja (Chieng and
Williams 1998
; Paxinos and Watson 1986
). Also in
some slices, the major islands of Calleja could be readily
distinguished, and in these cases, recordings were confined to an area
lateral to this structure. MSNs were visually distinguished from
interneurons in the NAc by the comparative size of their somata
(Chieng and Williams 1998
) using a fixed stage upright
microscope (Carl Zeiss Instruments, Germany) equipped with differential
interference contrast optics and infrared illumination (DIC-IR)
(Dodt and Zieglgansberger 1990
; Miller et al.
1997
; Svoboda et al. 1999
). In addition,
electrophysiological criteria (resting membrane potentials =
75
to
85 mV; absence of the H current, and absence of spontaneous
firing) were also used to distinguish these neurons (Chieng and
Williams 1998
; Uchimura et al. 1990
).
Whole cell recording
Whole cell patch-clamp recordings of NAc neurons were
performed using methods adapted from those described previously
(Lupica 1995; Miller et al.
1997
). Signals were acquired using an Axoclamp-2A, or an
Axopatch 200A amplifier (Axon Instruments, Burlingame, CA) and
electrodes pulled from thick-walled borosilicate capillary tubing (0.75 mm ID, 1.5 mm OD, Sutter Instrument, Novato, CA). Voltage or current
steps, used for monitoring series resistance, and for construction of
current-voltage (I-V) curves, were generated using a
Master-8 (AMPI, Jerusalem, Israel). Series resistance was monitored
continuously using small (10 mV), hyperpolarizing voltage steps (200 ms), and only cells demonstrating <20 M
series resistance were used
in these experiments. In most cases, the series resistance did not
change appreciably during the recording period. However, when the
series resistance increased, there was a noticeable decrease in whole
cell conductance and a sudden and sustained decrease in the holding
current. When this occurred the cell was not used in further analyses.
Excitatory postsynaptic currents (EPSCs) were recorded using whole cell
electrodes (5-7 M) filled with the following solution (in mM):
125.0 K+-gluconate, 10.0 KCl, 10.0 HEPES, 1.0 EGTA, 0.1 CaCl2, 2.0 Mg2+-ATP, and 0.2 Na+-GTP;
adjusted to pH 7.2-7.4 with 1 M KOH (270-280 mosM). During EPSC
recordings, neurons were voltage clamped at -80 to -90 mV, and
GABAA inhibitory postsynaptic currents (IPSCs)
were blocked by adding picrotoxin (100 µM) to the ACSF. IPSCs were
recorded in cells voltage clamped at
70 to
90 mV using whole cell
electrodes filled with the following solution (in mM): 125.0 CsCl, 10.0 HEPES, 1.0 EGTA, 0.1 CaCl2, 2.0 Mg2+-ATP, and 0.2 Na+-GTP
and the quaternary lidocaine derivative QX-314, 2, pH 7.2-7.4. During
IPSC recordings, glutamatergic EPSCs were blocked by adding the
glutamate receptor antagonists 6,7-dinitroquinoxaline-2,3-dione (DNQX,
10 µM) and D-(-)-2-amino-5-phosphonopentanoic acid (APV, 40 µM) to the ACSF.
Miniature IPSCs and EPSCs (mIPSCs, mEPSCs) were amplified 5- to
100-fold, filtered at 1-3 kHz and either recorded to videotape or
directly to the hard drive of a personal computer for later analysis.
Epochs of 1-3 min of data were digitized at 4-10 kHz using a National
Instruments (Austin, TX) Lab PC 1200 A/D converter and the Strathclyde
electrophysiology software package (courtesy of Dr. John Dempster,
Strathclyde University, Glasgow, UK,
http://innovol.sibs.strath.ac.uk/physpharm). The frequency, amplitudes,
and kinetic properties of these currents were then analyzed using the
Mini Analysis software package (v4.3, Synaptosoft, Leonia, NJ,
http://www.synaptosoft.com). Average mIPSCs/mEPSCs were generated by
aligning individual events by rise time, and a peak to decay single
exponential fit was applied to each average using the formula
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Evoked postsynaptic currents were generated using a bipolar tungsten
stimulating electrode placed within 100-150 µm of the recording
electrode. Whole cell access was monitored using voltage (or current)
step pulses (10-20 mV, 200 ms) delivered after each stimulus using
the Master-8 pulse generator. Stimulation (0.1-ms pulse duration) was
delivered at 15- to 30-s intervals using an optically isolated constant
current unit (AMPI) and the timer. Paired-pulse stimulation was
performed by delivering the same stimulus at either 150 ms (IPSC) or
100 ms (EPSC) inter-pulse intervals. In each experiment, stimulation
intensity was adjusted to evoke a submaximal response (50-400 µA).
Analyses of drug effects on evoked synaptic responses, membrane
potential, and input resistance were performed using PC-based software
(Neuropro, R. C. Electronics, Goleta, CA, or the Strathclyde
program, WCP v3.05).
Chemicals
Drugs were obtained from the following sources: tetrodotoxin (TTX), 6,7-dinitroquinoxaline-2,3-dione (DNQX), picrotoxin, ruthenium red, bicuculline methiodide, dopamine, [Tyr-D-Ala2, N-CH3-Phe4, Gly-ol-enkephalin] (DAMGO), and D-Ala2-methionine2-enkephalinamide (DALA), naloxone (Sigma, St. Louis, MO); APV, (RS)-baclofen, and WIN 55,212-2 (Tocris Cookson, Ballwin, MO). SR141716A was obtained from the National Institute on Drug Abuse drug-supply system. WIN 55,212-2 and SR141716A were prepared as concentrated (10 mM) stock solutions in DMSO. Final (bath) concentrations were <0.01% DMSO. All drugs were made up at either 50 or 100 times the desired final concentration in deionized water and then added to the flow of the superfusion medium using a calibrated syringe pump (Razel Scientific Instruments, Stamford, CT).
Statistical analysis
Group data are presented as the mean ± SE in all cases. Drug-induced changes in cumulative mEPSC and mIPSC amplitude and inter-event interval distributions were analyzed for statistical significance using the Kolmogorov-Smirnov test (Mini Analysis v4.3), with a conservative critical probability level of P < 0.01. All other statistical tests, including t-tests and ANOVAs, were performed using a critical probability of P < 0.05 (Prism version 3.0, GraphPad Software, San Diego, CA). Post hoc analysis (Newman-Keuls test) was performed only when an ANOVA yielded a significant (P < 0.05) main effect.
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RESULTS |
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Classification of neurons
Previous studies have demonstrated that the GABAergic MSNs are the
most numerous neurons in the NAc and that these cells can be
electrophysiologically distinguished from the smaller population of
interneurons (Chieng and Williams 1998; Uchimura
et al. 1990
). All of the neurons included in this study were
classified as MSNs based on the lack of spontaneous firing, relatively
hyperpolarized resting membrane potentials (
75 to -85 mV), and the
absence of a membrane sag associated with the
hyperpolarization-activated cation current
(Ih). The MSNs were further
distinguished from interneurons because their somata are much smaller
when visualized using DIC-IR microscopy (Chieng and
Williams 1998
).
Effects of WIN 55,212-2 on postsynaptic properties of NAc neurons
Although there is ample evidence to suggest that CB1 receptors are
localized to presynaptic terminals within the CNS, several studies have demonstrated postsynaptic effects of cannabinoids on a
variety of membrane conductances (Deadwyler et al. 1995; Mackie et al. 1995
; Schweitzer 2000
). To
determine whether cannabinoids alter the postsynaptic properties of
MSNs, we examined the effect of the cannabinoid agonist WIN 55,212-2 on
the passive membrane properties of these cells in current clamp. Under
control conditions, the mean resting membrane potential of these cells
(n = 11) was
83.5 ± 2.6 mV and the membrane
input resistance was 187 ± 25 M
. However, superfusion of WIN
55,212-2 (1 µM) for a period of time sufficient to achieve maximal
effects on synaptic responses (see following text) did not
significantly affect these parameters (resting membrane potential,
85.1 ± 2.7 mV, input resistance, 183 ± 16 M
,
P > 0.05, paired Student's 2-tailed
t-test). Furthermore, in cells voltage-clamped at
60 mV,
WIN 55,212-2 had no effect on either the steady-state conductance,
measured using a series of hyperpolarizing voltage steps
(n = 4; Fig. 1), or on
holding current (control, 60 ± 14 pA, WIN 55,212-2, 69 ± 9 pA, n = 4, P > 0.05, 2-tailed
Student's paired t-test). Thus WIN 55,212-2 did not alter
either the resting membrane properties, or whole cell conductance in
these MSNs.
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CB1 and µ-opioid modulation of evoked EPSCs in NAc neurons
Previous studies have demonstrated that CBs inhibit glutamatergic
transmission in several brain regions, including the hippocampus (Misner and Sullivan 1999; Shen et al.
1996
), cerebellum (Takahashi and Linden 2000
),
and substantia nigra (Szabo et al. 2000
). Thus to
determine whether glutamatergic inputs to the NAc were modulated by
cannabinoid receptors we isolated evoked EPSCs (evEPSCs) by electrically stimulating the NAc under conditions where
GABAA-receptor activity was eliminated by
addition of picrotoxin (100 µM). As shown in Fig.
2, the inward currents elicited under
these conditions were largely mediated by
non-N-methyl-D-aspartate (NMDA) glutamate receptors because they were nearly eliminated (12 ± 2% of
control, n = 5) by 10 µM DNQX. The µ-opioid agonist
DAMGO (1 µM) reversibly inhibited the evEPSCs (64 ± 7% of
control, P < 0.05, n = 8, paired t-test; Fig. 2). However, in contrast to the opioid effect,
the cannabinoid agonist WIN 55,212-2 (1 µM) caused a smaller
inhibition of these evEPSCs (Fig. 2, 82 ± 6% of control,
P < 0.05, n = 10, paired
t-test), that was only partially blocked by the CB1
antagonist SR141716A (Rinaldi-Carmona et al. 1994
) (1 µM, 93 ± 5% of control, P > 0.05, n = 4; paired t-test). Together, these data
suggest that µ-opioid receptors inhibit glutamatergic synaptic
transmission in the NAc to a greater extent than CB1 receptors.
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Because paired-pulse facilitation is a presynaptic phenomenon
(Mennerick and Zorumski 1995), pharmacological agents
that alter this process are thought to act presynaptically
(Chieng and Williams 1998
; Jiang et al.
2000
). To determine whether the inhibition of evoked EPSCs by
either CB1 or µ-opioid receptors was due to a presynaptic
interaction, we examined their effects on paired evEPSCs using
identical stimuli, delivered at a 100-ms inter-pulse interval. The
control average ratio of the second response to the first (EPSC2/EPSC1)
was 1.13 ± 0.05 (n = 9; Fig.
3). At the end of a 10- to 15-min
application of WIN 55,212-2 (1 µM), this ratio was not significantly
changed (1.15 ± 0.05, P = 0.65, 2-tailed paired
Student's t-test; Fig. 3). Similarly, the µ agonist DAMGO (1 µM) did not significantly effect the paired-pulse ratio (control, 1.18 ± 0.04; DAMGO, 1.17 ± 0.04, n = 9, P = 0.89, paired Student's t-test; Fig. 3).
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To determine whether the lack of changes in paired-pulse ratios was characteristic of our slice preparation, we also examined the effects of the GABAB receptor agonist, baclofen, and dopamine on these paired evEPSC responses. In direct contrast to the effects of the cannabinoid and the opioid agonist, both baclofen (30 µM; Fig. 3) and dopamine (30 µM) reversibly and significantly increased the paired evEPSC ratio (ratio values: control = 1.21 ± 0.11, baclofen = 1.45 ± 0.13, P = 0.03, n = 5; control = 1.26 ± 0.09, dopamine = 1.48 ± 0.13, wash = 1.12 ± 0.11, P = 0.02, n = 5; repeated-measures ANOVA and Newman-Keuls post hoc test) Thus both baclofen and dopamine increased paired evEPSC ratios, whereas the cannabinoid and opioid agonists had no effect on this parameter.
CB1 and µ-opioid modulation of mEPSCs in NAc neurons
The preceding results suggest that both WIN 55,212-2 and DAMGO did
not inhibit excitatory transmission in NAc MSNs through a presynaptic
mechanism. However, previous work has suggested that DAMGO acts both
pre- and postsynaptically to alter glutamatergic transmission in these
cells (Martin et al. 1997), and the mechanism of the
cannabinoid effect is unknown. Thus to explore these possibilities further, we examined the effects of WIN 55,212-2 and DAMGO on miniature, action potential-independent EPSCs (mEPSCs) in the presence
of the Na+ channel blocker TTX (500 nM).
Miniature EPSCs were recorded in MSNs voltage clamped at
80 to
90
mV using whole cell electrodes containing
K+-gluconate, in ACSF containing picrotoxin (100 µM). These responses are thought to reflect the quantal release of
glutamate acting at non-NMDA receptors (Nicola and Malenka
1997
). The mEPSCs recorded under these conditions
(n = 10) exhibited an average frequency of 1.7 ± 0.2 Hz, an average amplitude of 22.9 ± 1.6 pA, a decay time
constant of 8.3 ± 0.4 ms, and were greatly reduced in frequency and amplitude by DNQX (10 µM; Fig. 5A). These data
therefore suggest that the majority of these events were mediated by
glutamate activating non-NMDA receptors. The cannabinoid agonist WIN
55,212-2 (1 µM, n = 6) produced a small but
significant reduction in the average amplitude (paired Student's
t-test, P = 0.01, Fig.
4) of these mEPSCs but did not
significantly alter the frequency (Student's paired t-test,
P = 0.09) or the average decay time constant (control, 7.7 ± 0.3 ms, WIN 55,212-2, 8.9 ± 0.7 ms, P = 0.07, paired Student's t-test) of these events. In
contrast, the µ-opioid agonist DAMGO (1 µM, n = 4)
significantly reduced the mean mEPSC frequency (P < 0.05, paired Student's t-test, Fig.
5) but did not alter mean mEPSC amplitude
(P > 0.05, paired Student's t-test, Fig.
5) or the average decay time constant (control, 9.4 ± 0.6 ms,
DAMGO, 10 ± 0.5 ms, P = 0.19, paired Student's
t-test). In addition, the effect of DAMGO on mEPSC frequency
was reversed by application of the opioid receptor antagonist, naloxone
(NLX, 5 µM, Fig. 5). These data suggest that the cannabinoid receptor
agonist WIN 55,212-2 produced a small reduction of glutamatergic
transmission through a postsynaptic mechanism while DAMGO likely acted
via a presynaptic mechanism.
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CB1 and opioid receptor effects on evoked IPSCs in NAc neurons
There is ample evidence that cannabinoids inhibit GABAergic
neurotransmission in other brain areas (Chan et al.
1998; Hoffman and Lupica 2000
; Szabo et
al. 1999
; Vaughan et al. 1999
, 2000
). Therefore
we examined whether GABAergic IPSCs were also modulated by cannabinoids
in the NAc and compared these effects to those of opioid receptor
activation. In these experiments, MSNs were voltage-clamped at
80 to
90 mV using whole cell electrodes containing CsCl, with APV (40 µM)
and DNQX (10 µM) included in the superfusion medium to block
glutamatergic transmission. Under these conditions, electrical
stimulation of the slice produced large inward currents that were
completely blocked by the GABAA antagonist
bicuculline methiodide (BMI, 20 µM, Fig.
6). As shown in Fig. 6A, WIN
55,212-2 (1 µM) caused a time-dependent decrease in the evIPSC
amplitude that was maximal at 8-10 min into the agonist application.
This inhibition of evIPSCs was seen in every MSN examined. As we have previously reported, the highly lipophilic nature of cannabinoid ligands results in a relatively long period of time to achieve equilibrium in the tissue, and precludes washout during these experiments (Hoffman and Lupica 2000
). Therefore to
ensure cannabinoid receptor mediation of these effects, we pretreated
several slices with the selective CB1 receptor antagonist SR141716A (1 µM) for 10-15 min prior to WIN 55,212-2 application. As previously
reported in the hippocampus (Hoffman and Lupica 2000
),
the CB1 antagonist had no effect by itself on the evIPSC (93 ± 8% of control, P > 0.05, 1 sample t-test)
but completely blocked the expected inhibition during WIN 55,212-2 application (Fig. 6, B and C). In a further attempt to establish whether the effects of WIN 55,212-2 were receptor
mediated and to compare its effects in the NAc with those in other
systems, we constructed a concentration-response curve using the
cumulative administration of WIN 55,212-2 (10 nM-5 µM) while
measuring evIPSCs in NAc neurons. As shown in Fig.
7, WIN 55,212-2 inhibited the evoked IPSC
in a concentration-dependent fashion, with an estimated
EC50 of 123 nM.
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In contrast to the robust inhibition of evIPSCs by the cannabinoid
agonist, the µ-opioid agonist DAMGO (1 µM) had no effect on these
responses (Fig. 6, A and B), suggesting that
these receptors do not modulate GABA release onto MSNs in the NAc.
Because DAMGO is a highly selective µ-opioid receptor agonist
(Goldstein and Naidu 1989), we also examined the
possible modulation of evIPSCs with the nonselective µ/
opioid
agonist DALA. In contrast to the effects of DAMGO, DALA (5 µM)
significantly reduced evIPSCs recorded in NAc MSNs (69 ± 8% of
control, P < 0.05, repeated measures ANOVA,
n = 11, Fig. 8). However,
it was found that, unlike the activation of CB1 receptors in which
virtually all evIPSCs were inhibited, only a fraction (~50%) of
these responses were significantly inhibited by DALA. Thus the effects
of DALA on evIPSCs could be further divided into two groups: those in
which DALA had a large effect (45 ± 6% of control,
n = 5) and those in which DALA had a very small effect
(89 ± 8% of control, n = 6). Therefore it appears that while virtually all evIPSCs were inhibited by WIN 55,212-2, the effects of opioid receptor activation were much more
variable, ranging from no significant response for the selective µ agonist, DAMGO, to a significant inhibition of evIPSCs with the
nonselective opioid agonist DALA in a subpopulation of MSNs.
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In an effort to establish whether the observed inhibition of GABAergic transmission reflected pre- or postsynaptic actions, we examined the effects of WIN 55,212-2 and DALA on paired evIPSCs elicited with stimuli separated by 150 ms. In the absence of the agonist, the second evIPSC of the pair was inhibited relative to the first, indicating a modest paired-pulse depression (IPSC2/ISPC1 = 0.84 ± 0.05, n = 10). However, during WIN 55,212-2 (1 µM) application, IPSC2 was, on average, significantly larger than evIPSC1 (IPSC2/IPSC1 = 1.2 ± 0.11, P < 0.05 vs. control, paired Student's t-test; Fig. 9), suggesting a presynaptic site of WIN 55,212-2 action. Similarly, the opioid agonist DALA (5 µM) significantly increased the paired evIPSC ratio to 1.5 ± 0.25 (n = 5, P < 0.05, repeated-measures ANOVA, Newman-Keuls) in the group of neurons that was highly sensitive to DALA, and this effect was reversed by NLX (5 µM; ratio = 0.84 ± 0.17).
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CB1 receptor effects on miniature IPSCs in NAc neurons
The results described above demonstrate that, in contrast to its
small effect on EPSCs, WIN 55,212-2 robustly inhibited evIPSCs in NAc
MSNs. Moreover the paired-pulse experiments demonstrate a likely
presynaptic mechanism for this inhibition. To more fully explore the
mechanism of the cannabinoid effects on
GABAA-mediated synaptic transmission and to
further examine the possible role that postsynaptic actions may play,
we studied the effects of WIN 55,212-2 on TTX-insensitive mIPSCs. These
experiments were conducted in MSNs voltage clamped at 80 to
90 mV,
using whole cell electrodes containing CsCl and in the presence of
glutamate receptor antagonists (40 µM APV and 10 µM DNQX) in the
ACSF. Under these conditions, small spontaneous inward currents were
observed that were completely blocked by addition of BMI (20 µM; Fig.
10B). In contrast to other
classes of neurons routinely studied in our laboratory (e.g.,
hippocampal pyramidal neurons) (Hoffman and Lupica
2000
), the frequency of the spontaneously occurring currents in
MSNs in the absence of TTX was very low (0.7 ± 0.2 Hz,
n = 6). Because of this, the application of TTX (0.5 µM) caused only a small change in the frequency of these events
(0.5 ± 0.1 Hz, Fig. 10, A and C). Despite
the low frequency of occurrence, the CB1 agonist WIN 55,212-2 (1 µM)
significantly reduced the frequency of mIPSCs in two of six cells
(P < 0.01, K-S test; Fig. 10B), and these
effects were reversed by the CB1 antagonist SR 141617A (1 µM; Fig.
10B). However, when the effects of the CB1 agonist on all
cells were averaged, this effect was not statistically significant (repeated measures ANOVA, P > 0.05). Similarly, there
was no significant effect of the agonist on mIPSC amplitude (Fig.
10C).
|
Because we were concerned that the low frequency of mIPSCs in
these MSNs might affect the reliability of our analysis, we attempted
to increase their number with the bath application of the polyvalent
cation ruthenium red. Ruthenium red has been shown to increase the
frequency of GABAergic mIPSCs, in the presence of TTX, through a direct
extracellular interaction with secretory mechanisms
(Sciancalepore et al. 1998; Trudeau et al.
1996
). In addition, ruthenium red has been shown to block
voltage-dependent calcium channels, eliminating them as a possible site
of action (Cibulsky and Sather 1999
). In these
experiments, mIPSCs were first monitored in the presence of TTX.
Ruthenium red (200 µM) was then applied for
10 min prior to
application of WIN 55,212-2. Ruthenium red produced a large eightfold
increase in the frequency of mIPSCs (mIPSC frequency: TTX = 0.37 ± 0.1 Hz; ruthenium red = 3.1 ± 0.3 Hz,
n = 4) that was maximal within 6 min of beginning its
application (Fig. 11B). The
average amplitude of the mIPSCs was also increased by ruthenium red,
although not to a statistically significant level (mIPSC amplitude:
TTX = 17.0 ± 2.1 pA, ruthenium red = 19.4 ± 1.8 pA, ANOVA, P > 0.05). The time constant for the decay
of the averaged mIPSCs was unaffected by ruthenium red (decay time
constants: TTX = 51.2 ± 2.9 ms, ruthenium red = 53.4 ± 3.1 ms), and these mIPSCs were completely eliminated by
BMI (20 µM, n = 4, Fig. 11B), confirming
that they were mediated by the activation of
GABAA receptors. WIN 55,212-2 (1 µM) caused no
change in the average amplitudes of these mIPSCs (Fig. 11C)
nor did it change the time constant for the mean mIPSC decay (50 ± 2.8 ms). However, WIN 55,212-2 caused a small (20-25%) but
significant reduction in the average frequency of mIPSCs 12-18 min
after beginning its application (Fig. 11, A and
C; n = 4; repeated-measures ANOVA
P < 0.01; P < 0.01 Kolmogorov-Smirnov
test).
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DISCUSSION |
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The present study reports several novel findings regarding the
actions of cannabinoids and opioids on the physiology of MSNs located
in the shell region of the NAc. First, the cannabinoid agonist WIN
55,212-2 did not affect postsynaptic passive membrane properties of
MSNs in vitro. Second, glutamatergic synaptic inputs to NAc neurons
were only marginally inhibited by WIN 55,212-2 but robustly inhibited
by the µ-opioid agonist DAMGO. Third, GABAergic synaptic responses
were consistently inhibited by WIN 55,212-2 in all cells, inhibited by
the µ/ opioid agonist DALA in some cells, and completely
insensitive to the µ-opioid receptor agonist DAMGO. Together, these
data demonstrate the differential modulation of excitatory and
inhibitory synaptic transmission by cannabinoids in the NAc and
distinguishes these actions from those of opioids.
A common action of many drugs of abuse, including cannabinoids, is to
increase the levels of dopamine in the NAc (Chen et al.
1990; Di Chiara and Imperato 1988
). Since
dopamine directly inhibits both excitatory and inhibitory amino
acid-mediated synaptic transmission in the NAc (Harvey and Lacey
1996
, 1997
; Nicola and Malenka 1997
;
Pennartz et al. 1992
), it is likely that this mechanism plays an important role in regulating network activity, and hence reward, in this structure. Therefore since activation of cannabinoid receptors in the VTA (French 1997
) increases dopamine
levels in the NAc (Chen et al. 1990
), it is also
possible that this mechanism contributes to the rewarding properties of
marijuana. On the other hand, several drugs of abuse, including opioids
(Chieng and Williams 1998
; Martin et al.
1997
), cocaine (Nicola et al. 1996
), amphetamine (Nicola and Malenka 1997
; Nicola et al.
1996
), and PCP (Carlezon and Wise 1996
) have
been shown to reduce amino acid-mediated synaptic transmission by
direct actions in the NAc. Moreover all of these drugs are
self-administered by animals into the NAc (Carlezon and Wise
1996
; McBride et al. 1999
), again suggesting
that their rewarding properties are related, at least in part, to their
direct actions on NAc synapses. In contrast to these more commonly
studied drugs of abuse, direct effects of cannabinoids within the NAc have not been demonstrated until now. However, the moderately high
level of CB1 receptor expression in this brain region (Herkenham et al. 1991
; Tsou et al. 1998
) indicates that
these receptors might mediate the effects of cannabinoids in the NAc.
Cannabinoid agonists have been shown to modulate postsynaptic
K+ channels in hippocampal pyramidal neurons
(Deadwyler et al. 1995; Schweitzer 2000
)
and in cellular expression systems (Mackie et al. 1995
).
However, our data suggest that inwardly rectifying K+ channels were not modulated in MSNs by WIN
55,212-2 because membrane potential, holding current, input resistance,
and the conductance of MSNs were unchanged. This is consistent with
results in periacqueductal gray (PAG) neurons (Vaughan et al.
2000
) and hippocampal interneurons (unpublished data). However,
because we did not attempt to isolate specific voltage-dependent
postsynaptic conductances, we cannot exclude possible effects of
cannabinoids on these ion channels (Deadwyler et al.
1995
; Schweitzer 2000
).
Cannabinoid and opioid modulation of glutamatergic transmission
Cannabinoids have been shown to inhibit glutamate release in the
hippocampus (Misner and Sullivan 1999; Shen et
al. 1996
), cerebellum (Takahashi and Linden
2000
), and substantia nigra (Szabo et al. 2000
).
Therefore we examined the effects of WIN 55,212-2 on pharmacologically
isolated glutamatergic EPSCs in MSNs (Martin et al.
1997
; Pennartz et al. 1991
, 1992
). These
experiments demonstrated that, in contrast to the robust
cannabinoid-mediated inhibition of glutamatergic transmission observed
in other brain regions, evEPSCs were only modestly inhibited by the
cannabinoid agonist WIN 55,212-2 in the NAc, and these effects were
only partially reversed by the CB1 antagonist SR141716A. However, the
µ-opioid agonist DAMGO reduced these responses to a larger degree
(Martin et al. 1997
). These data therefore suggest that
excitatory inputs to MSNs were more strongly inhibited by µ-opioids
than by cannabinoids in MSNs.
To determine whether the cannabinoid- and opioid-mediated inhibition of
EPSCs reflected actions at pre- or postsynaptic sites, we examined the
effects of WIN 55,212-2 and DAMGO on the paired-pulse facilitation of
evEPSCs (Harvey and Lacey 1996). We found that neither
WIN 55,212-2 nor DAMGO modulated evEPSC paired-pulse ratios despite the
pronounced facilitation of these responses by dopamine and baclofen
(also see Harvey and Lacey 1996
). This suggested that,
in contrast to dopamine and baclofen, the inhibitory effects of DAMGO
or WIN 55,212-2 on evEPSCs were not presynaptic. To more fully explore
this possibility, we used a more powerful analysis of TTX-resistant
mEPSCs. These quantal release events are a very sensitive measure of
the locus of a drug's effect because changes in mEPSC amplitudes are
associated with a postsynaptic site of drug action, whereas changes in
mEPSC frequency are likely due to interaction with a presynaptic site
(Cohen et al. 1992
; Lupica 1995
;
Thompson et al. 1993
). In the present study, WIN
55,212-2 decreased mEPSC amplitude, without altering mEPSC frequency,
whereas DAMGO reduced mEPSC frequency but did not affect mEPSC
amplitude. Thus these data suggested that WIN 55,212-2 acted
postsynaptically, whereas DAMGO acted presynaptically to inhibit
glutamatergic transmission. Given these results, it is unclear why
paired-pulse facilitation was unaffected by the opioid. However,
because NMDA receptor function is known to be augmented by both DAMGO
(Martin et al. 1997
) and repetitive stimulation
(Pennartz et al. 1991
) in MSNs, it is possible that this
postsynaptic change obscured DAMGO's presynaptic actions, which were
clearly seen in the mEPSC experiments. This idea is supported by data
demonstrating that almost no NMDA component contributes to mEPSCs
(Nicola and Malenka 1997
; present study). However,
despite this disparity, both the mEPSC and the paired evEPSC
experiments suggest that the small effect of WIN 55,212-2 on
glutamatergic transmission likely occurred through a postsynaptic interaction, whereas the effects of DAMGO were likely presynaptic on
mEPSCs, and both pre- and postsynaptic on the paired evEPSC responses
(Martin et al. 1997
). The mechanism of this apparent postsynaptic effect of WIN 55,212-2 will require further investigation.
Cannabinoid and opioid modulation of inhibitory transmission
In direct contrast to its modest effects on evEPSCs, the
cannabinoid agonist more strongly inhibited
GABAA-mediated evIPSCs in NAc MSNs. The effect of
WIN 55,212-2 was concentration-dependent (EC50 = 123 nM) and completely antagonized by the selective CB1 antagonist
SR141716A (Rinaldi-Carmona et al. 1994). Furthermore the
estimated potency of WIN 55,212-2 was similar to that reported for
evIPSCs in hippocampal pyramidal neurons (138 nM) (Hoffman and
Lupica 2000
). The antagonist also had no effect alone on
evIPSCs, suggesting that endogenous cannabinoids do not modulate
synaptic transmission in NAc brain slices under the present conditions (Hoffman and Lupica 2000
; Katona et al.
1999
).
In comparison with the consistent effect of the cannabinoid on GABA
release onto MSNs, the effects of the opioid agonists were much more
variable. The selective µ-opioid receptor agonist DAMGO had no effect
on the evIPSCs, and the nonselective µ/-opioid agonist DALA
inhibited evIPSCs in only a subset of the MSNs examined (~50%).
Since DALA does not have significant activity at
-opioid receptors
(Goldstein and Naidu 1989
) and its effects were reversed by NLX, we conclude that these actions were on a subset of inhibitory terminals that express
-opioid receptors. A previous study has shown
inhibition of evoked synaptic potentials in NAc MSNs by µ-,
-, and
-opioid receptors (Yuan et al. 1992
). However, it was
unclear whether these depolarizing responses were composed of EPSPs,
IPSPs, or both. Furthermore in that study, only a small number of the
neurons demonstrated clear hyperpolarizing IPSPs that were inhibited by
selective µ- or
-opioid agonists. In contrast, the present data
support the more recent observations that
-opioid receptors are more
prominently represented on GABAergic terminals in the NAc than are
µ-opioid receptors, which are largely associated with postsynaptic
GABAergic cellular profiles (Svingos et al. 1997
, 1998
).
Our data also suggest that
-opioid receptors may only be found on a
subset of the inhibitory terminals making contact with MSNs. Thus
unlike the effect of CB1 receptor activation, which inhibited GABA
release onto all MSNs,
-opioid receptors seemed to more selectively
regulate GABA release onto only a subset of these neurons.
Unlike its effect on glutamatergic transmission in the present study
the cannabinoid agonist, WIN 55,212-2, likely reduced GABAergic
transmission in the NAc through, exclusively, a presynaptic action.
This was supported by data showing a switch from paired-pulse depression to facilitation of evIPSCs during CB1 receptor (and opioid
receptor) activation and a complete absence of WIN 55,212-2 effects on
mIPSC amplitudes or decay kinetics. In these experiments, and in a
previous study (Nicola and Malenka 1997), the frequency of mIPSCs was found to be very low. This was somewhat surprising because the fast spiking GABAergic interneurons that are thought to
provide the major inhibitory input to MSNs in the striatum (and
probably the NAc; see following text) are spontaneously active in vivo
and in slice preparations (Kawaguchi et al. 1995
;
Koós and Tepper 1999
) This may be explained by the
fact that these previous experiments were conducted at higher
temperatures (35°C), whereas those in the present study, and in the
study by Nicola and Malenka (1997)
, were conducted at
room temperature, which tends to decrease the number of spontaneously
active cells. In an attempt to mitigate possible deleterious effects of
a small sample size on our analysis of mIPSCs, we also examined these events in the presence of ruthenium red, which enhances mIPSC frequency
by a direct interaction with the transmitter release machinery
(Sciancalepore et al. 1998
; Trudeau et al.
1996
). This experiment also demonstrated that WIN 55,212-2 had
no effect on mIPSC amplitude or kinetics and only a small effect on
mIPSC frequency. Taken together, these results strongly suggest a
presynaptic site of action for the cannabinoid in the NAc; an effect
that is also found in other brain areas (Chan et al.
1998
; Hoffman and Lupica 2000
; Szabo et
al. 1998
; Vaughan et al. 1999
, 2000
).
Functional implications for the modulation of NAc output by cannabinoids
Prior descriptions of striatal/NAc physiology have attributed
GABAergic inhibition of MSNs to synaptic interactions among these cells
through an extensive network of axon collaterals (Park et al.
1980; Wilson and Groves 1980
). However, more
recent data appear to refute this concept, suggesting instead that the
major source of inhibitory GABAergic input to MSNs is derived from a much smaller population of striatal interneurons (Jaeger et al. 1994
; Kawaguchi et al. 1995
; Koós
and Tepper 1999
). In particular, one class of these intrinsic
interneurons has been shown to express mRNAs for glutamic acid
decarboxylase (GAD67) and the CB1 receptor (Hohmann and
Herkenham 2000
). Assuming that this relationship is found in
the ventral striatum/NAc, it is likely that the presynaptic inhibition
of GABA release by WIN 55,212-2 seen in the present study was due to
activation of CB1 receptors located on the terminals of these
interneurons. Furthermore because these interneurons receive
substantial cortical glutamatergic input (Bennett and Bolam
1994
; Pennartz and Kitai 1991
), they represent a
critical link in mediating cortico-accumbens feedforward inhibition of medium spiny projection neuron output. Therefore one of the roles of
CB1 receptors in the NAc might be to dampen this feedforward inhibition, thereby disinhibiting MSNs, making them more responsive to
excitatory cortical input and increasing NAc output to the ventral
pallidum and VTA. In contrast, our data demonstrating a more
circumscribed modulation of GABA release onto MSNs by opioid receptors
suggest that this system may have a more limited role in the
disinhibition of these NAc projection neurons that may allow certain
groups of cells to function as discrete NAc output pathways
(Groenewegen et al. 1999
; Pennartz et al.
1994
).
The role of the NAc in drug reward, abuse, and addiction has been the
subject of intensive investigation for many decades. While it is clear
that the rewarding properties of these drugs are associated with
changes in NAc activity, there is still considerable debate as to the
specific mechanisms involved. Increases in NAc dopamine levels are a
useful neurochemical index of drug reward but do not fully account for
the complex processing of fast synaptic activity by this neuromodulator
in the NAc. Moreover because both glutamatergic and GABAergic inputs to
MSNs are directly inhibited by dopamine, as well as by drugs of abuse,
it is likely that these effects contribute to the rewarding properties
of these drugs. Thus a demonstration of direct actions at NAc synapses
provides a useful context in which to consider the rewarding properties of a drug, either apart from, or in addition to, its effects on NAc
dopamine levels. Indeed it is unlikely that the effects of WIN 55,212-2 in the present study were due to an increase in dopamine levels because
a recent voltammetric study demonstrated that this agonist did not
increase dopamine levels in NAc slices (Szabo et al.
1999). Furthermore we did not see the robust inhibition of
glutamatergic EPSCs that would be expected with elevated dopamine levels (Harvey and Lacey 1996
). However, because
cannabinoids increase dopamine levels in the NAc in vivo (Chen
et al. 1990
), it is likely that when systemically administered
they would indirectly inhibit glutamatergic synapses through this
mechanism (Harvey and Lacey 1996
). Despite the
complexity of these direct and indirect actions of drugs of abuse
on amino acid-mediated synaptic processes in the NAc, it is likely that
the modulation of the population activity of MSNs through these
synaptic mechanisms will contribute to the rewarding and addictive
properties of these drugs in the intact organism.
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ACKNOWLEDGMENTS |
---|
The authors thank Dr. Roy A. Wise for helpful comments on the manuscript.
This work was supported by the National Institute on Drug Abuse.
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FOOTNOTES |
---|
Present address and address for reprint requests: C. R. Lupica, Dept. of Pharmacology, University of Arizona Health Sciences Center, 1501 N. Campbell Ave., Tucson, AZ 85724-5050 (E-mail: crlupica{at}emailarizona.edu).
Received 15 June 2000; accepted in final form 15 September 2000.
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REFERENCES |
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