Download Increased Mesolimbic GABA Concentration Blocks Heroin Self

0022-3565/00/2942-0613$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics
JPET 294:613–619, 2000 /2551/834824
Vol. 294, No. 2
Printed in U.S.A.
Increased Mesolimbic GABA Concentration Blocks Heroin SelfAdministration in the Rat1
ZHENG-XIONG XI and ELLIOT A. STEIN
Departments of Cellular Biology, Neurobiology and Anatomy (Z.-X.X., E.A.S.), Psychiatry and Behavioral Medicine (E.A.S.), Pharmacology and
Toxicology (E.A.S.), and The Biophysics Research Institute (E.A.S.), Medical College of Wisconsin, Milwaukee, Wisconsin
Accepted for publication April 4, 2000
This paper is available online at http://www.jpet.org
Heroin is the most rapidly acting and most abused of the
opiates. Unfortunately, its high abuse liability is not matched
by effective pharmacological therapy. Currently, the most
effective treatment strategy is opiate replacement therapy
with methadone or its derivative, l-␣-acetylmethadone. However, these drugs are accompanied by their own dependence
liability, emphasizing the need for new treatment strategies
based on reducing opiate reinforcement.
The mesocorticolimbic dopamine (DA) system, which originates in the ventral tegmental area (VTA) and projects rostrally
to the nucleus accumbens (NAcc) and the medial prefrontal
cortex, has been consistently demonstrated to play a critical role
in mediating opiate reinforcement (Bardo, 1998). Although the
mechanisms underlying this reinforcement are still incompletely understood, the current hypothesis is that opiates inhibit GABAergic cells to disinhibit mesocorticolimbic system
DA neurons (Johnson and North, 1992a). Several lines of evidence support this hypothesis. For example, systemic or VTA
Received for publication January 28, 2000
1
This investigation was supported in part by National Institute on Drug
Abuse Grant DA09465 and the Schering Plough Research Foundation.
not the GABAA antagonist bicuculline. Similarly, coadministration
of heroin with aminooxy-acetic acid (1– 4 mg/kg) or ethanolamineO-sulfate (50 –100 mg/kg), two reversible GABA transaminase
inhibitors, dose dependently reduced heroin reinforcement. Coadministration of (⫾)-nipecotic acid (0.1–5 mg/kg) with heroin, or
intra-VTA or -ventral pallidum pretreatment with (⫾)-nipecotic acid
(10 ␮g) or NO-711 (2 ␮g), two GABA uptake inhibitors, significantly
increased heroin SA behavior, an effect also blocked by systemic
2-hydroxysaclofen, but not bicuculline. Taken together, these experiments, for the first time, demonstrate that pharmacological
elevation of mesolimbic GABA concentration blocks heroin reinforcement by activating GABAB receptors, supporting the
GABAergic hypothesis of opiate reinforcement and the incorporation of GABA agents in opiate abuse treatment.
microiontophoretic morphine increases the firing rate of dopaminergic neurons and inhibits the firing rate of inhibitory interneurons (Gysling and Wang, 1983; Johnson and North,
1992a). Microdialysis and electrochemical studies demonstrate
an increase in NAcc DA release after heroin administration
(Spanagel et al., 1990; Xi et al., 1998). Furthermore, opiate
␮-receptors are predominantly located on VTA GABAergic interneurons (Dilts and Kalivas, 1989), and morphine presynaptically inhibits ␥-aminobutyric acid (GABA) release within the
medial portion of the rat midbrain (Renno et al., 1992). We (Xi
and Stein, 1998, 1999) have previously demonstrated that pharmacological down-regulation of VTA DA neuronal activity and
NAcc DA release after administration of ␬-opiate agonists or the
GABAB agonist baclofen reduces heroin reinforcement.
Thus, although the mechanisms of opiate action within the
VTA are emerging, it is as yet unclear whether similar actions of opiates also occur in the NAcc. Experimental evidence has suggested both a DA-dependent and DA-independent mechanism processing opiate reinforcement within the
NAcc. For example, whereas 6-hydroxydopamine lesions
within the NAcc can disrupt morphine self-administration
ABBREVIATIONS: DA, dopamine; GABA, ␥-aminobutyric acid; GABAT, GABA transaminase; GVG, ␥-vinyl-GABA; 2-OH-saclofen, 2-hydroxysaclofen; SA, self-administration; CPP, conditioned place preference; VP, ventral pallidum; VTA, ventral tegmental area; NAcc, nucleus
accumbens; AOAA, aminooxy-acetic acid; EOS, ethanolamine-O-sulfate; NipA, (⫾)-nipecotic acid; i.c., intracranial.
613
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 7, 2017
ABSTRACT
Opiate reinforcement has been hypothesized to be mediated by
an inhibition of mesolimbic ␥-aminobutyric acid (GABA) release
that subsequently disinhibits ventral tegmental area (VTA) dopamine neurons. In support of this hypothesis, this study demonstrates that when administered directly into the lateral ventricle,
the VTA, or the ventral pallidum, but not the nucleus accumbens,
␥-vinyl-GABA (GVG, an irreversible GABA-transaminase inhibitor,
20 –50 ␮g) dose dependently blocked heroin (0.06 mg/kg) selfadministration (SA), as assessed by an increase in heroin SA at
low doses of GVG and an initial increase followed 1 to 2 h later by
a blockade of heroin SA at higher GVG doses. This effect lasted 3
to 5 days. In drug-naı̈ve rats, intra-VTA GVG pretreatment also
prevented or delayed acquisition of heroin SA for 2 days. This
GVG effect was prevented or reversed by systemic or intra-VTA
pretreatment with the GABAB antagonist 2-hydroxysaclofen, but
614
Xi et al.
Materials and Methods
Surgical Preparation. Male Sprague-Dawley rats (Sasco, Madison, WI) weighing 250 to 350 g at the time of surgery were individually housed and maintained on a 12-h light/dark cycle (lights on at
8:00 PM) with free access to food and water. One hundred rats were
subdivided into five heroin SA groups: ␥-vinyl-GABA (GVG; n ⫽ 44),
aminooxy-acetic acid (AOAA; n ⫽ 16), ethanolamine-O-sulfate (EOS;
n ⫽ 8), (⫾)-nipecotic acid (NipA; n ⫽ 28), and a saline substitution
group (n ⫽ 4). Based on the specific experiment, some rats received
more than one drug, and their numbers are represented in more than
one of the above groups. Under sodium pentobarbital anesthesia (60
mg/kg i.p.), heroin SA rats were implanted with a chronic silicone
rubber jugular catheter that passed s.c. to terminate on a head
assembly. To observe the effects of central receptor modulation on
heroin SA, 96 rats were also implanted with 30-gauge stainless steel
guide cannula bilaterally into the lateral ventricles (coordinates: 0.8
mm posterior to bregma, 1.6 mm lateral to midline, and 4.2 mm
ventral to the surface of the cortex), the VTA (4.8 mm posterior to
bregma, 1.0 mm lateral to midline, and 7.7 mm ventral to the surface
of the cortex), the NAcc (1.6 mm anterior to bregma, 1.6 mm lateral
to midline, and 7.2–7.4 mm ventral to the surface of the cortex), or
the VP (0.3 mm anterior to bregma, 2.5 mm lateral to midline, and
7.8 mm ventral to the surface of the cortex). Three days were allowed
for recovery from surgery before SA training.
Heroin-SA Procedure. Operant boxes (30 ⫻ 40 ⫻ 60 cm),
equipped with a lever mounted on one side wall 5 cm above the cage
floor, were housed in sound- and light-attenuated chambers. The i.v.
catheter was connected to a syringe pump (Razel, Stamford, CT)
through polyethylene tubing and a liquid commutator. Each lever
press delivered an infusion of heroin (approximately 100 ␮l) over a
10-s period. Depending on the experiment, heroin (0.06 mg/kg) or
heroin plus a GABA agent dissolved in sterile saline was administered per lever press. A 60-W white light located above the chamber
was simultaneously illuminated with each drug infusion. Each SA
session lasted 4 h, and each rat was tested for 5 to 9 days on a fixed
ratio 1 schedule.
The effects of the GABA agents on SA behavior (GVG, 20 –50 ␮g
i.c.v., VTA, NAcc, or VP; AOAA, 1– 4 mg/kg i.v.; EOS, 50 –100 mg/kg
i.v.; NipA, 0.1–5 mg/kg i.v. or 10 –20 ␮g bilaterally directly into the
VTA or VP) were assessed after stable SA behavior was established
(within ⫾5% of the mean responses for 3 days of heroin alone training). Additionally, to observe the effects of GVG on heroin SA acquisition, a group of drug-naı̈ve rats received GVG before heroin exposure. All GABA agents were purchased from Research Biochemicals
International (Natick, MA) and dissolved fresh each day in sterile
saline. Heroin was donated by the Resource Technology Branch,
National Institute on Drug Abuse (Bethesda, MD). All injections into
the lateral ventricles, the VTA, the NAcc, or the VP were delivered in
a volume of 1 or 2 ␮l over 1 or 2 min, respectively. Intracranial (i.c.)
drug injection doses were divided evenly into each hemisphere and
are reported as total amount administered.
Statistic Analyses. All data are presented as mean ⫾ S.E. Student’s t test and/or two-way ANOVAs were used to assess drug effect
significance. A P ⬍ .05 was used throughout.
Histology. On completion of each SA experiment, rats were
deeply anesthetized with pentobarbital and transcardially perfused
with phosphate-buffered saline followed by 10% formalin. Brains
were sectioned at 40 ␮m, and cannulas tips verified histologically.
Only rats with properly located cannula were included in subsequent
behavioral data analyses.
Results
Heroin SA Behavior. Typically, rats rapidly learned the
operant task and reliably self-administered heroin after 2 to
3 days of training. Four rats with unstable SA behavior or
misplaced or blocked i.c. cannula were eliminated from the
study and excluded from data analysis. Although SA rates
and interinjection intervals varied somewhat across rats and
sessions, the pattern of responses and the mean SA rate
across sessions were very stable over time (Fig. 1, heroin
control curve).
Effects of GABAT Inhibitors on Heroin SA. When microinjected into the lateral ventricles (i.c.v.), the irreversible
GABAT inhibitor GVG (20 ␮g, n ⫽ 6) significantly increased
heroin SA behavior for about 24 h, whereas a higher dose (50
␮g, n ⫽ 4) first decreased and then completely blocked SA
behavior 2 h after GVG administration, an effect that lasted
for about 3 days (Fig. 1, A and B). In an identical manner,
pretreatment with GVG (20 –50 ␮g) into the VTA also dose
dependently inhibited heroin reinforcement. Although a significant increase in heroin SA was seen after low-dose (20 ␮g)
administration, the pattern of operant responding after highdose GVG (50 ␮g) was more complex, with an increase in
lever pressing in the first 1 to 2 h followed by a virtual
cessation during the remaining hours of the 4-h session (data
not shown). A similar pattern of responding was seen after
saline substitution during heroin SA (Fig. 1, A and B). This
GVG blockade lasted 3 to 4 days after a single VTA injection
(Fig. 2A). Intra-VP administration of the same doses of GVG
also altered heroin SA with the same dose- and time-dependent patterns as intra-VTA administration (Fig. 3, A and B).
In contrast, the 50-␮g dose of GVG injected bilaterally into
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 7, 2017
(SA) (Smith et al., 1985), systemic or intra-accumbal administration of DA antagonists does not alter i.v. heroin SA
(Ettenberg et al., 1982). Destruction of NAcc presynaptic DA
terminals selectively attenuates cocaine, but not heroin, SA
(Pettit et al., 1984). Opiates, when systemically self-administered (Chang et al., 1997; Lee et al., 1999), or locally administered into the NAcc (Hakan and Henriksen, 1989), significantly inhibit the spontaneous firing of NAcc neurons.
Because NAcc efferent projections are mostly GABAergic
(Groenewegen and Russchen, 1984; Kalivas et al., 1993) and
terminate principally in the ventral pallidum (VP) and the
VTA (Mogenson and Nielson, 1983), two critical areas in
mediating opiate reinforcement, we hypothesize that, similar
to the VTA, opiates also inhibit NAcc GABAergic projection
cells that ultimately disinhibit postsynaptic VP neurons and
VTA DA neurons. Such a mechanism may, in part, explain
the proposed DA-independent opiate reward mechanism.
To test the hypothesis that opiate reinforcement is mediated, at least in part, by inhibiting both VTA and NAcc
GABAergic cells, this study examined the effects of elevated
endogenous mesolimbic GABA concentration on heroin SA.
After being released into the synaptic cleft where it activates
specific GABA receptor subtypes, GABA is taken up into
presynaptic neuronal terminals and glial cells by GABA uptake carriers (Krogsgaard-Larsen and Johnston, 1975),
where it is further catabolized by the enzyme GABAtransaminase (GABAT) (Sabers and Gram, 1992). Thus, the
strategy used in this study was to elevate GABA concentration by administering GABAT or GABA uptake inhibitors
systemically or directly into the lateral ventricles, VTA,
NAcc, or VP either before or during heroin SA. Our results
support the GABAergic hypothesis of opiate reinforcement by
demonstrating dose- and time-dependent SA inhibition after
administration of GABA-enhancing drugs.
Vol. 294
2000
Mesolimbic GABA Inhibits Heroin SA
615
Fig. 1. Intracerebroventricular administration of GVG dose
dependently blocks heroin (0.06 mg/kg/injection) reinforcement. A, time-dependent GVG effect across experimental
days. After 3 days of heroin SA, GVG was administered on
day 4 (arrow). The low dose of GVG (F, 20 ␮g, n ⫽ 6)
significantly increased heroin SA, suggesting a partial reinforcement blockade, whereas the high dose of GVG (E, 50
␮g, n ⫽ 4) almost completely blocked heroin SA 2 h after
GVG administration. The low-dose GVG effect lasted about
2 days, whereas the high-dose GVG effect lasted at least 3
days. Saline substitution on days 5 and 6 (open arrow) did
not maintain SA behavior in rats previously trained to SA
heroin (䉫, n ⫽ 4). In this and all subsequent figures, the
heroin (0.06 mg/kg/injection) alone group (f) represents all
rats (n ⫽ 30) that received 6 days of heroin SA training. B,
time course of the day 4 GVG experiment plotted as a
function of session time. **P ⬍ .01, compared with heroin
alone group.
NipA, a GABA uptake inhibitor, increased heroin SA (Fig.
6A). Similarly, microinjections of NipA into the VTA (10 ␮g)
or the VP (10 ␮g) consistently increased operant responding
for heroin (Fig. 6B). Another selective GABA uptake inhibitor, NO-711, microinjected into the VTA (2 ␮g), also significantly increased heroin SA behavior (Fig. 6B). The effect of
NipA was selectively attenuated by systemic injection of
2-OH-saclofen (2 mg/kg, n ⫽ 8), but not bicuculline (0.1
mg/kg, n ⫽ 8) (Fig. 7). Systemic 2-OH-saclofen or bicuculline
administered alone did not significantly affect SA behavior.
Discussion
This study demonstrates, for the first time, that elevating
synaptic GABA levels by direct i.c. administration of the
GABAT inhibitors GVG, AOAA, or EOS, or the GABA uptake
inhibitors NipA or NO-711, dose dependently reduces heroin
reinforcement (defined as an increase in SA at low doses and
a time-dependent SA blockade at higher treatment doses,
independent of nonspecific effects on locomotion), and prevents or delays acquisition of heroin SA. This effect was seen
with i.c.v., VTA, or VP injections, but not after NAcc administration. The inhibitory effects of intra-VTA GVG or systemic NipA were blocked or reversed by the GABAB antagonist 2-OH-saclofen, but not the GABAA antagonist
bicuculline, suggesting an effect mediated by GABAB receptors.
VTA GABAergic Mechanism of Opiate Reinforcement. Two types of neurons, primary dopaminergic projection neurons and secondary GABAergic inhibitory interneurons, have been identified within the VTA. Both the intrinsic
VTA GABAergic interneurons and GABAergic axon terminals from the NAcc synapse onto VTA DA neurons (Johnson
and North, 1992b; Kalivas et al., 1993). Systemic or iontophoretic administration of morphine increases the firing rate
of the DA neurons by binding to ␮-opiate receptors located
predominantly on and inhibiting GABAergic interneurons
(Matthews and German, 1984; Dilts and Kalivas, 1989; Johnson and North, 1992a).
This VTA GABA microcircuitry is supported by several
behavioral and neurochemical studies: 1) opiates inhibit
GABA release from the rat midbrain (Renno et al., 1992), 2)
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 7, 2017
the NAcc had no significant effect on heroin SA (Fig. 4).
Likewise, intra-NAcc administration of another GABAT inhibitor, AOAA (2 ␮g), also had no effect on heroin SA. AOAA
did, however, significantly increase heroin SA when microinjected into the VTA (Fig. 4).
To determine whether GVG could also interfere with the
acquisition of heroin SA, GVG was administered into the
VTA (50 ␮g, n ⫽ 8) before the first day of heroin exposure. A
single injection was able to delay heroin SA acquisition for 2
days in heroin-naı̈ve rats (Fig. 2B). Thereafter, these rats
gradually began responding at rates comparable with rats
receiving heroin alone. After SA was established, a second
GVG injection was given (day 7) and induced a marked
increase in SA. This large burst in operant behavior mainly
occurred within the first 1 to 2 h of the 4-h SA session,
followed once again by complete SA blockade the following
day.
To determine the receptor mechanisms mediating this
GVG effect, the GABAA and GABAB receptor antagonists
bicuculline and 2-hydroxysaclofen (2-OH-saclofen), respectively, were administered systemically in separate groups of
rats. After stable SA behavior, 2-OH-saclofen (2 mg/kg), but
not bicuculline (0.1 mg/kg), pretreatment blocked the SA
inhibition induced by intra-VTA GVG administration. In contrast, neither 2-OH-saclofen nor bicuculline alone significantly altered heroin SA (days 4 and 6, respectively in Fig.
2C). Consistent with this observation, intra-VTA 2-OHsaclofen (2 ␮g) administered three days after GVG-induced
SA blockade also reversed the SA inhibition (n ⫽ 4), with
behavioral rates increasing from 1.8 ⫾ 0.2 to 9.5 ⫾ 2.3 SA/4
h (data not shown).
The effects of two reversible GABAT inhibitors, AOAA
(1– 4 mg/kg) and EOS (50 –100 mg/kg), on heroin SA were
examined in two additional groups of rats. Coadministration
of either drug with heroin dose dependently blocked heroin
reinforcement, manifest as a compensatory increase in SA at
the lower doses and blockade of SA at the higher doses of
each drug (Fig. 5, A and B). Similarly, intra-VTA microinjection of AOAA (2 ␮g) also significantly increased heroin SA
(Fig. 4).
Effects of GABA Uptake Inhibitors on Heroin SA.
When coadministered with heroin, all doses (0.1–5 mg/kg) of
616
Xi et al.
intra-VTA microinjections of the GABAB agonist baclofen
block opiate-induced DA release in the VTA (Klitenick et al.,
1992) and the NAcc (Kalivas et al., 1990), and 3) intra-VTA
baclofen blocks morphine-induced conditioned place preference (CPP; Tsuji et al., 1996) and heroin SA behavior (Xi and
Stein, 1999). Similarly, baclofen alone inhibits firing of VTA
DA neurons (Johnson and North, 1992a) and reduces VTA
and NAcc DA release (Klitenick et al., 1992; Xi and Stein,
1998), suggesting that VTA GABAB receptors play a critical
role in mediating endogenous GABA modulation of VTA DA
neurons. In contrast, GABAA receptors are located mainly on
VTA interneurons (Dilts and Kalivas, 1989), with their activation disinhibiting VTA DA cells, thereby increasing NAcc
DA release (Xi and Stein, 1998).
By directly manipulating mesolimbic GABA concentration,
both by inhibiting the GABA degradation enzyme GABAT or
by inhibiting GABA uptake, the current data support the
above opiate reinforcement hypothesis. In addition, intraVTA GVG pretreatment not only blocked heroin-reinforced
SA but also prevented or delayed acquisition of heroin SA in
drug-naı̈ve rats, and did so for up to 4 days after a single
injection. This effect was blocked or reversed by i.v. or intraVTA pretreatment with the GABAB antagonist 2-OHsaclofen, but not by the GABAA antagonist bicuculline, suggesting that the GVG effect was mediated by increased
GABA concentration acting on VTA GABAB receptors. These
data are consistent with our previous reports demonstrating
that the selective GABAB agonist baclofen, but not the
GABAA agonist muscimol, blocks heroin reinforcement (Xi
and Stein, 1998, 1999). Because administration of GVG,
AOAA, and EOS had no significant effects on spontaneous
locomotor behavior, these data suggest a specific GABA-induced reinforcement blockade, rather than a nonspecific effect on motor behavior.
The relatively long duration inhibitory effect of GVG administration on heroin SA is consistent with the drug’s irreversible effect on GABA transaminase, requiring de novo
synthesis of new enzyme. For example, GVG dose dependently increases brain GABA concentration for more than
48 h (Jung et al., 1977; Qume and Fowler, 1996). It should be
noted, however, that GVG also inhibits GABA uptake, which
may have contributed to its strong inhibitory effect on heroin
reinforcement (Christensen et al., 1991; Jolkkonen et al.,
1992).
Consistent with the ability of GVG to reduce heroin reinforcement, GVG also is known to decrease DA release in the
NAcc and the striatum (Morgan and Dewey, 1998) and can
significantly antagonize cocaine-induced CPP (Morgan et al.,
1997) and locomotor behavioral sensitization (Dewey et al.,
1997). Taken together, these data suggest that heroin and
cocaine share a common mesolimbic GABAergic reinforcement mechanism.
Finally, the reversible GABAT inhibitors, AOAA and EOS,
also dose dependently reduced heroin reinforcement. Low
doses of both drugs increased, whereas high doses completely
blocked heroin SA behavior. However, in contrast to the
central effect of GVG, intra-VTA administration of AOAA
only increased heroin SA, an effect that lasted for less than
24 h; complete SA blockade was never seen with any dose
tested. This latter observation is consistent with the pharmacological properties of AOAA acting as a competitive, reversible GABAT inhibitor (Qume and Fowler, 1996). Similarly, coadministration of heroin with NipA, a GABA uptake
inhibitor (Krogsgaard-Larsen and Johnston, 1975), significantly increased SA within a wide dose range, while administration of NipA or NO-711 into either the VTA or VP also
only increased heroin SA, suggesting partial heroin reinforcement reduction. Incomplete blockade of heroin reinforcement by NipA, even at a dose 50 times higher than its
minimal effective dose, may suggest that GABA uptake
mechanisms play a secondary role to GABAT degradation in
synaptic GABA removal. Taken together, however, these
data strongly support a role for both VTA and NAcc GABA in
heroin reinforcement.
NAcc GABAergic Mechanism of Opiate Reinforcement. Several lines of evidence suggest that, in addition to
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 7, 2017
Fig. 2. Bilateral intra-VTA GVG pretreatment dose dependently blocked
heroin reinforcement. A, after heroin SA training for 3 days, GVG was
administered on day 4 (arrow). Low-dose GVG (E, 20 ␮g, n ⫽ 7) increased
SA rate, whereas high-dose GVG (‚, 50 ␮g, n ⫽ 8) decreased SA behavior.
Both effects lasted 3 to 4 days. B, intra-VTA pretreatment with GVG (‚,
50 ␮g) in drug-naı̈ve rats delayed the acquisition of heroin SA for 4 days,
compared with behavioral acquisition in the heroin alone control group. A
second injection of GVG on day 7 of SA training dramatically increased
SA, followed by complete SA blockade on the following day (n ⫽ 8). The
increased SA response was manifest within the first few hours of the 4-h
session on day 7, followed by virtual operant cessation. C, the GABAB
antagonist 2-OH-saclofen (Sac; 2 mg/kg, i.v.), but not the GABAA antagonist (⫺)-bicuculline (Bic; 0.1 mg/kg, i.v.), blocked the GVG-induced inhibition of heroin SA (F, n ⫽ 4). While neither 2-OH-saclofen nor bicuculline alone altered heroin SA (days 4 and 6, respectively), 2-OHsaclofen on day 5 prevented the GVG effect, whereas bicuculline on day 7
did not. The solid curve (f, n ⫽ 30) in panels A, B, and C shows the heroin
alone SA group. *P ⬍ .05, **P ⬍ .01, compared with either the same day
of the heroin only group or day 3 of the experimental group.
Vol. 294
2000
Mesolimbic GABA Inhibits Heroin SA
617
Fig. 3. GVG pretreatment into the VP dose dependently
blocked heroin SA behavior. A, time-dependent GVG effect
across experimental days. After 3 days of heroin SA training, GVG was administered on day 4 (arrow). The low GVG
dose (F, 20 ␮g, n ⫽ 5) significantly increased heroin SA,
suggesting a partial reinforcement blockade, whereas the
high dose of GVG (E, 50 ␮g, n ⫽ 3) almost completely
blocked SA behavior. Both dose effects lasted at least 3
days. B, time course of day 4 GVG administration on heroin
SA plotted as a function of time within session. The high
dose caused a biphasic increase in responding that, after
2 h, completely suppressed responding. The solid curve (f,
n ⫽ 30) shows the heroin SA control group. **P ⬍ .01,
compared with heroin alone group.
Fig. 5. Dose-dependent effects of AOAA (A) and EOS (B) on
heroin SA. A, time course of AOAA effects during 4-h SA
sessions plotted as time within session. At 1 mg/kg (F, n ⫽
8), AOAA significantly increased heroin SA behavior,
whereas 2 mg/kg (䉫, n ⫽ 8) induced a compensatory increase in SA within the first half-hour that progressed to a
time-dependent decrease in responding; 4 mg/kg (E, n ⫽ 8)
AOAA almost completely blocked heroin SA during the
entire session. All dose effects were significant after 2 h
when compared with heroin control group. B, time course of
EOS effects during 4-h SA sessions. The low dose of EOS
(䉫, 50 mg/kg, n ⫽ 6) increased SA, whereas the high dose
(E, 100 mg/kg, n ⫽ 6) significantly reduced SA behavior.
The solid curve (f) shows the heroin alone control.
the VTA, the NAcc is also involved in processing opiate
reinforcement. When assessed by either SA or CPP, intraaccumbal injections of opiates exert reinforcing (Van der
Kooy et al., 1982; Goeders et al., 1984) effects that can be
blocked by intra-NAcc administration of opiate antagonists
(Vaccarino et al., 1985). While specific neurochemical mechanisms are still poorly understood, it has been proposed that
opiate-induced NAcc DA release plays a critical role, possibly
by inhibiting NAcc GABAergic efferent cells (Swerdlow et al.,
1990; Bourdelais and Kalivas, 1992). However, several pieces
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 7, 2017
Fig. 4. Pretreatment with the GABAT inhibitors GVG and
AOAA into the NAcc had no effect on heroin SA. A, time
course of GVG (F, 50 ␮g, n ⫽ 9) and AOAA (E, 2 ␮g, n ⫽ 4)
on heroin SA plotted during daily 4-h training sessions. B,
total SA responding within 4-h sessions after NAcc GVG or
AOAA administration had no effect on heroin SA. In contrast, intra-VTA administration of AOAA (2 ␮g, n ⫽ 3)
significantly increased heroin SA, suggesting partial reinforcement blockade. The solid curve (f, n ⫽ 30) shows the
heroin SA control group. Numbers in parentheses represent
the number of rats in each group. *P ⬍ .05, compared with
heroin alone group.
618
Xi et al.
Vol. 294
Fig. 6. Effects of the GABA uptake inhibitors NipA and
NO-711 on heroin SA. A, coadministration of NipA increased heroin SA. A shallow dose-response curve was seen
with an increase in SA rate observed at all doses, suggesting a partial reinforcement blockade. The solid and dashed
lines show the mean ⫾ S.E. heroin alone control level. B,
microinjections of NipA into the VTA (10 ␮g) or VP (10 ␮g)
significantly increased heroin SA. NO-711 (2 ␮g) injection
into the VTA also significantly increased heroin SA rate.
Numbers in parentheses represent the number of rats in
each group. *P ⬍ .05, **P ⬍ .01, compared with heroin
alone group.
of evidence conflict with this DA hypothesis. For example,
while 6-hydroxydopamine NAcc lesions have been reported to
disrupt opiate reward (Smith et al., 1985), systemic or intraaccumbal administration of DA antagonists does not alter
heroin SA (Ettenberg et al., 1982), and destruction of NAcc
presynaptic DA terminals selectively attenuates cocaine but
not heroin SA (Pettit et al., 1984). Electrophysiological studies have shown that local microiontophoretic application of
morphine into the NAcc (Hakan and Henriksen, 1989), or i.v.
SA of heroin (Chang et al., 1997; Lee et al., 1999), markedly
suppresses NAcc neuronal activity. These data suggest that
both a DA-dependent and a DA-independent mechanism may
exist within the NAcc to mediate opiate reinforcement. However, the precise circuitry of such a proposed DA-independent
reinforcement mechanism is still unclear.
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 7, 2017
Fig. 7. The GABAB antagonist 2-OH-saclofen (Sac; 2 mg/kg, i.v.), but not
the GABAA antagonist bicuculline (Bic; 0.1 mg/kg, i.v.), reduced the
NipA-induced SA increase. However, neither 2-OH-saclofen nor bicuculline alone altered heroin SA. Numbers in parentheses represent the
number of rats per group. *P ⬍ .05, **P ⬍ .01, compared with heroin
alone group. #P ⬍ .05, compared with NipA and heroin coadministration
group.
Based on the VTA GABAergic hypothesis, we now hypothesize that opiates inhibit NAcc GABAergic projection neurons by acting on both VTA DA projection neurons and directly within the NAcc, the latter comprising a non-DAdependent reinforcement mechanism. Thus, only interfering
with the former may incompletely block opiate reinforcement
by leaving the latter non-DA mechanism intact. Because the
major NAcc efferents are GABAergic and project both to the
VP and back to the VTA (Groenewegen and Russchen, 1984;
Kalivas et al., 1993), opiate inhibition of NAcc GABA neurons
can disinhibit both the target VP neurons and the VTA DA
projection neurons. As such, the former action will produce a
DA-independent opiate reinforcement, whereas the latter
will potentiate the mesolimbic DA-dependent mechanism in
a positive feedback manner. In the present experiment, GVG
administered into the VP or VTA, two main NAcc GABAergic
projection areas, dose dependently reduced heroin reinforcement, supporting this GABAergic disinhibitory hypothesis.
VP GABAergic Mechanism of Opiate Reinforcement.
Although the VP serves as a major anatomical target of NAcc
efferent projections and provides inputs to the medial prefrontal cortex, amygdala, and lateral hypothalamus, systems
subserving drug-taking behavior (Groenewegen et al., 1993),
its role in mediating opiate reward is less well understood.
Pallidal GABAergic fibers project back to the NAcc and the
VTA to form a complex local modulatory circuit (Kalivas et
al., 1993; Churchill and Kalivas, 1994). In this study, intraVTA administration of GVG, AOAA, or NipA significantly
reduced heroin reinforcement, suggesting that the VP projections to VTA DA neurons play an important role in mediating
opiate reinforcement. In contrast, intra-NAcc administration
of GVG or AOAA had no significant effect on heroin SA,
consistent with our previous report demonstrating that intra-NAcc injection of the GABAB agonist baclofen had no
effect on heroin reinforcement (Xi and Stein, 1999). In addition, ibotenic acid lesions of VP neurons block both heroin
and cocaine SA behavior, supporting a role for the VP in drug
reinforcement (Hubner and Koob, 1990).
In summary, we have demonstrated that elevation of endogenous GABA concentration by either GABAT inhibitors
or GABA uptake inhibitors consistently blocks heroin SA
behavior. These data provide, for the first time, direct evi-
2000
dence to support the GABAergic hypothesis of opiate reinforcement and suggest that GABA enhancers, such as GVG
and NipA, may be effective in treating opiate addiction.
Acknowledgments
We thank Zachary M. Pruhs, Gregory J. Gosline, and Jun Tang
who participated in this project during summer studies in the lab.
References
619
Kalivas PW, Duffy P and Eberhardt H (1990) Modulation of A10 dopamine neurons
by ␥-aminobutyric acid agonists. J Pharmacol Exp Ther 253:858 – 866.
Klitenick MA, DeWitte P and Kalivas PW (1992) Regulation of somatodendritic
dopamine release in the ventral tegmental area by opioids and GABA: An in vivo
microdialysis study. J Neurosci 12:2623–2632.
Krogsgaard-Larsen P and Johnston GA (1975) Inhibition of GABA uptake in rat
brain slices by nipecotic acid, various isoxazoles and related compounds. J Neurochem 25:797– 801.
Lee RS, Criado JR, Koob GF and Henriksen SJ (1999) Cellular responses of nucleus
accumbens neurons to opiate-seeking behavior: I. Sustained responding during
heroin self-administration. Synapse 33:49 –58.
Matthews RT and German DC (1984) Electrophysiological evidence for excitation of
rat ventral tegmental area dopamine neurons by morphine. Neuroscience 11:617–
625.
Mogenson GJ and Nielson MA (1983) Evidence that an accumbens to subpallidal
GABAergic projection contributes to locomotor activity. Brain Res Bull 11:309 –
314.
Morgan AE and Dewey SL (1998) Effects of pharmacologic increases in brain GABA
levels on cocaine-induced changes in extracellular dopamine. Synapse 28:60 – 65.
Morgan AE, Porter SP, Clarkson FA, Kuhar MJ, Carroll FI, Volkow ND, Fowler JS
and Dewey SL (1997) A direct approach for attenuating cocaine’s effects on extracellular dopamine: Targeting the dopamine transporter. Synapse 26:423– 427.
Pettit HO, Ettenberg A, Bloom FE and Koob GF (1984) Destruction of dopamine in
the nucleus accumbens selectively attenuates cocaine but not heroin selfadministration in rats. Psychopharmacology 84:167–173.
Qume M and Fowler LJ (1996) Effects of chronic oral treatment with GABAT
inhibitors on the GABA system in brain, liver, and plasma of the rat. Biochem
Pharmacol 52:1355–1363.
Renno WM, Mullett MA and Beitz AJ (1992) Systemic morphine reduces GABA
release in the lateral but not the medial portion of the midbrain periaqueductal
gray of the rat. Brain Res 594:221–223.
Sabers A and Gram L (1992) Pharmacology of vigabatrin. Pharmacol Toxicol 70:
237–240.
Smith JE, Guerin GF, Co C, Barr TS and Lane JD (1985) Effects of 6-OHDA lesions
of the central medial nucleus accumbens on rat intravenous morphine selfadministration. Pharmacol Biochem Behav 23:843– 849.
Spanagel R, Herz A and Shippenberg TS (1990) The effects of opioid peptides on
dopamine release in the nucleus accumbens: An in vivo microdialysis study.
J Neurochem 55:1734 –1740.
Swerdlow NR, Braff DL and Geyer MA (1990) GABAergic projection from nucleus
accumbens to ventral pallidum mediates dopamine-induced sensorimotor gating
deficits of acoustic startle in rats. Brain Res 532:145–150.
Tsuji M, Nakagawa Y, Ishibashi Y, Yoshii T, Takashima T, Shimada M and Suzuki
T (1996) Activation of ventral tegmental GABAB receptors inhibits morphineinduced place preference in rats. Eur J Pharmacol 313:169 –173.
Vaccarino FJ, Bloom FE and Koob GF (1985) Blockade of nucleus accumbens opiate
receptors attenuates intravenous heroin reward in the rat. Psychopharmacology
86:37– 42.
Van der Kooy D, Mucha RF, O’Shaughnessy M and Bucenieks P (1982) Reinforcing
effects of brain microinjections of morphine revealed by conditioned place preference. Brain Res 243:107–117.
Xi Z-X, Fuller SA and Stein EA (1998) Dopamine release in the nucleus accumbens
during heroin self-administration is modulated by kappa opioid receptors: An in
vivo fast-cyclic voltammetry study. J Pharmacol Exp Ther 284:151–161.
Xi Z-X and Stein EA (1998) Nucleus accumbens dopamine release modulation by
mesolimbic GABAA receptors: An in vivo electrochemical study. Brain Res 798:
156 –165.
Xi Z-X and Stein EA (1999) Baclofen inhibits heroin self-administration and mesolimbic dopamine release. J Pharmacol Exp Ther 290:1369 –1374.
Send reprint requests to: Elliot A. Stein, Ph.D., Department of Psychiatry,
8701 Watertown Plank Rd., Milwaukee, WI 53226. E-mail: [email protected]
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 7, 2017
Bardo MT (1998) Neuropharmacological mechanism of drug reward: Beyond dopamine in the nucleus accumbens. Crit Rev Neurobiol 12:37– 67.
Bourdelais A and Kalivas PW (1992) Apomorphine decreases extracellular GABA in
the ventral pallidum of rats with 6-OHDA lesions in the nucleus accumbens. Brain
Res 577:306 –311.
Chang JY, Zhang L, Janak PH and Woodward DJ (1997) Neuronal responses in
prefrontal cortex and nucleus accumbens during heroin self-administration in
freely moving rats. Brain Res 754:12–20.
Christensen H, Fykse EM and Fonnum F (1991) Inhibition of gamma-aminobutyrate
and glycine uptake into synaptic vesicles. Eur J Pharmacol 207:73–76.
Churchill L and Kalivas PW (1994) A topographically organized gammaaminobutyric acid projection from the ventral pallidum to the nucleus accumbens
in the rat. J Comp Neurol 345:579 –595.
Dewey SL, Chaurasia CS, Chen CE, Volkow ND, Clarkson FA, Porter SP, Straughter-Moore RM, Alexoff DL, Tedeschi D, Russo NB, Fowler JS and Brodie JD (1997)
GABAergic attenuation of cocaine-induced dopamine release and locomotor activity. Synapse 25:393–398.
Dilts RP and Kalivas PW (1989) Autoradiographic localization of ␮-opioid and
neurotensin receptor within the mesolimbic dopamine system. Brain Res 488:311–
327.
Ettenberg A, Pettit HO, Bloom FE and Koob GF (1982) Heroin and cocaine intravenous self-administration in rats: Mediation by separate neural systems. Psychopharmacology 78:204 –209.
Goeders NE, Lane JD and Smith JE (1984) Self-administration of methionine enkephalin into the nucleus accumbens. Pharmacol Biochem Behav 20:451– 455.
Groenewegen HJ, Berendse HW and Haber SN (1993) Organization of the output of
the ventral striatopallidal system in the rat: Ventral pallidal efferents. Neuroscience 57:113–142.
Groenewegen HJ and Russchen FT (1984) Organization of the efferent projections of
the nucleus accumbens to pallidal, hypothalamic, and mesencephalic structures: A
tracing and immunohistochemical study in the cat. J Comp Neurol 223:347–367.
Gysling K and Wang RY (1983) Morphine-induced activation of A10 dopamine
neurons in the rat. Brain Res 277:119 –127.
Hakan RL and Henriksen SJ (1989) Opiate influences on nucleus accumbens neuronal electrophysiology: Dopamine and non-dopamine mechanisms. J Neurosci
9:3538 –3546.
Hubner CB and Koob GF (1990) The ventral pallidum plays a role in mediating
cocaine and heroin self-administration in the rat. Brain Res 508:20 –29.
Johnson SW and North RA (1992a) Opioids excite dopamine neurons by hyperpolarization of local interneurons. J Neurosci 12:483– 488.
Johnson SW and North RA (1992b) Two types of neurons in the rat ventral tegmental
area and their synaptic input. J Physiol (Lond ) 450:455– 468.
Jolkkonen J, Mazurkiewicz M, Lahtinen H and Riekkinen P (1992): Acute effects of
gamma-vinyl GABA on the GABAergic system in rats as studied by microdialysis.
Eur J Pharmacol 229:269 –274.
Jung MJ, Lippert B, Metcalf BW, Bohlen P and Schechter PJ (1977) ␥-Vinyl -GABA
(4-amino-hex-5-enoic acid), a new selective irreversible inhibitor of GABA-T: Effects on brain GABA metabolism in mice. J Neurochem 29:797– 802.
Kalivas PW, Churchill L and Klitenick MA (1993) GABA and enkephalin projection
from the nucleus accumbens and ventral pallidum to the ventral tegmental area.
Neuroscience 57:1047–1060.
Mesolimbic GABA Inhibits Heroin SA