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Inactivation of the bed nucleus of the stria terminalis in an
animal model of relapse: effects on conditioned cue-induced
reinstatement and its enhancement by yohimbine
Deanne M. Buffalari and
Department of Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260, USA
Ronald E. See
Department of Neurosciences, Medical University of South Carolina, BSB416B, 173 Ashley
Avenue, Charleston, SC 29425, USA
Ronald E. See: seere@musc.edu
Abstract
Rationale—Drug-associated cues and stress increase craving and lead to greater risk of relapse in
abstinent drug users. Animal models of reinstatement of drug seeking have been utilized to study
the neural circuitry by which either drug-associated cues or stress exposure elicit drug seeking.
Recent evidence has shown a strong enhancing effect of yohimbine stress on subsequent cue-
elicited reinstatement; however, there has been no examination of the neural substrates of this
interactive effect.
Objectives—The current study examined whether inactivation of the bed nucleus of the stria
terminalis (BNST), an area previously implicated in stress activation of drug seeking, would affect
reinstatement of cocaine seeking caused by conditioned cues, yohimbine stress, or the combination
of these factors.
Methods—Male rats experienced daily IV cocaine self-administration, followed by extinction of
lever responding in the absence of cocaine-paired cues. Reinstatement of responding was
measured during presentation of cocaine-paired cues, following pretreatment with the
pharmacological stressor, yohimbine (2.5 mg/kg, IP), or the combination of cues and yohimbine.
Results—All three conditions led to reinstatement of cocaine seeking, with the highest
responding seen after the combination of cues and yohimbine. Reversible inactivation of the
BNST using the gamma-aminobutyric acid receptor agonists, baclofen+muscimol, significantly
reduced all three forms of reinstatement.
Conclusion—These results demonstrate a role for the BNST in cocaine seeking elicited by
cocaine-paired cues, and suggest the BNST as a key mediator for the interaction of stress and cues
for the reinstatement of cocaine seeking.
Keywords
Cocaine; Yohimbine; Reinstatement; Relapse; Bed nucleus stria terminalis; Norepinephrine;
Neurocircuitry; Stress; Conditioned; Self-administration
© Springer-Verlag 2010
Correspondence to: Ronald E. See, seere@musc.edu.
NIH Public Access
Author Manuscript
Psychopharmacology (Berl). Author manuscript; available in PMC 2011 July 8.
Published in final edited form as:
Psychopharmacology (Berl)
. 2011 January ; 213(1): 19–27. doi:10.1007/s00213-010-2008-3.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Introduction
Treatment of cocaine addiction is impeded by high rates of relapse to drug seeking and drug
taking in chronic users. Relapse can be triggered by many factors, including exposure to
drug-associated cues and contexts (such as drug paraphernalia or drug-associated
environments) or stressful life events that occur during periods of abstinence. Clinical
laboratory studies have shown that cocaine-associated stimuli increase craving in abstinent
users (Childress et al. 1993), as does exposure to stress-provoking stimuli (Sinha et al.
1999). The triggering of relapse in abstinent users has been modeled in animals using the
self-administration and reinstatement paradigm. Animals trained to self-administer cocaine
will reinstate responding on the previously drug-paired lever after exposure to cocaine-
associated cues (See 2002), stress (Shalev et al. 2000), or a priming dose of cocaine (de Wit
and Stewart 1981). The reinstatement model of relapse (Katz and Higgins 2003) has allowed
for the investigation of the neural circuitry underlying cocaine-seeking behavior triggered by
each of these factors. Evidence to date suggests that cues and stress share some of the same
neural pathways in mediating reinstatement of cocaine seeking, most notably the nucleus
accumbens core and the dorsomedial prefrontal cortex (Capriles et al. 2003; Fuchs et al.
2004a; McFarland et al. 2004; McLaughlin and See 2003). However, some structures (e.g.,
basolateral amygdala) have been found to be important for cue- but not footshock stress-
induced reinstatement of cocaine seeking (McFarland et al. 2004; Meil and See 1997).
While almost all previous studies of the triggering events that initiate reinstatement of drug
seeking have limited their focus to separate factors, we have recently demonstrated that
footshock or yohimbine stress activation potentiates conditioned cue-induced reinstatement
of cocaine seeking (Buffalari and See 2009a; Feltenstein and See 2006). The mechanisms by
which this interaction occurs likely involve a convergence of activity in the neural pathways
that reinitiate drug seeking. Among the structures that mediate drug seeking, the bed nucleus
of the stria terminalis (BNST) is a key component of the extended amygdala that may be a
critical point of convergence for cue and stress interactions. Studies of the BNST have
indicated that it plays a key role in addictive drug actions, including cocaine (Dumont et al.
2005; Kash et al. 2008). Inactivation of the BNST with sodium channel blockers (Erb and
Stewart 1999) or gamma-aminobutyric acid (GABA) receptor agonists (McFarland et al.
2004), as well as beta norepinephrine (NE) receptor antagonism (Leri et al. 2002), have been
shown to block reinstatement of cocaine seeking caused by acute footshock stress. However,
it is not yet known if the BNST plays a role in conditioned cue-induced reinstatement of
cocaine seeking, or reinstatement caused by other stressors.
Several recent studies have successfully used systemic injections of yohimbine to trigger
stress-induced reinstatement of drug seeking in rats (Banna et al. 2010; Bongiovanni and
See 2008; Feltenstein and See 2006; Shepard et al. 2004) and nonhuman primates (Lee et al.
2004), and elicit drug craving in human addicts (Stine et al. 2002). Yohimbine increases NE
in terminal regions via antagonism of α-2 NE receptors (Galvez et al. 1996; Tjurmina et al.
1999). However, nothing is yet known about the neural circuitry underlying reinstatement
caused by systemic yohimbine, or any other drugs that may act as stressors. Both
intermittent footshock (Galvez et al. 1996) and yohimbine (Forray et al. 1997) increase NE
in the amygdala, including the extended amygdala and BNST, and NE receptor blockade in
the BNST disrupts cocaine seeking caused by footshock stress (Leri et al. 2002), supporting
the possibility that stress activation via yohimbine may rely on intact BNST function.
Therefore, in the current study, we examined the role of the BNST in reinstatement of
cocaine seeking caused by yohimbine-induced stress alone, conditioned cues alone, or a
combination of yohimbine and cues in rats with a history of chronic cocaine self-
administration and extinction. Based on the reported role of the BNST in both stress
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activation and drug seeking, we predicted that the BNST would be critical for mediating
stress-induced reinstatement via yohimbine, as well as the enhancing effects of yohimbine
on conditioned cue-induced reinstatement of cocaine seeking.
Materials and methods
Subjects
Male Sprague–Dawley rats (initial weight 275–300 g; Charles River, Wilmington, MA,
USA) were individually housed in a temperature- and humidity-controlled vivarium on a
reverse 12-h light–dark cycle (lights on 6 PM–6 AM). Animals received water and standard
rat chow (Harlan, Indianapolis, IN, USA) ad libitum, with the exception of 2–3 days of food
restriction during initial cocaine self-administration (animals never received <10 g food/
day). Housing and care of the rats were carried out in accordance with the National Institute
of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23)
revised 1996.
Surgery
Rats received chronically indwelling catheters into the right jugular vein as previously
described (Fuchs et al. 2004b). Briefly, male rats were anesthetized using a mixture of
ketamine hydrochloride and xylazine (66 and 1.33 mg/kg, respectively, IP) followed by
equithesin (0.5 ml/kg, IP). Catheters (constructed using previously described methods;
Fuchs et al. 2004b) were inserted into the right jugular vein. To maintain patency, catheters
were flushed with heparin and cefazolin solutions daily. Immediately following catheter
surgery, animals were placed into a stereotaxic frame (Stoelting, Wood Dale, IL, USA).
Bilateral stainless steel guide cannulae (26 gauge; Plastics One, Inc.) were inserted dorsal to
the BNST (±3.5 M/L, −0.4 A/P, −4.6 D/V, 15° angle). Three small screws and cranioplastic
cement secured the guide cannulae to the skull. Stylets (Plastics One, Inc.) were placed into
the guide cannulae and catheter to prevent occlusions. To verify catheter patency, rats
occasionally received a 0.12-ml infusion of methohexital sodium (10.0 mg/ml IV; Eli Lilly
and Co., Indianapolis, IN, USA), a short-acting barbiturate that produces a rapid loss of
muscle tone when administered intravenously.
Cocaine self-administration
Rats self-administered cocaine (cocaine hydrochloride dissolved in 0.9% sterile saline;
cocaine provided by the National Institute on Drug Abuse, Research Triangle Park, NC,
USA) during daily 2-h sessions according to a fixed ratio-1 (FR 1) schedule of
reinforcement as previously described (Feltenstein et al. 2007b; Fuchs et al. 2004b). Briefly,
lever presses on the active lever resulted in cocaine infusions along with presentation of a
light+tone complex and were followed by a 20-s timeout. Inactive lever presses had no
consequences, but were recorded. Daily cocaine self-administration continued until each rat
had obtained the self-administration criterion of 10 sessions with at least 10 infusions per
session.
Extinction and reinstatement of cocaine seeking
Following chronic self-administration and before the first reinstatement test, rats underwent
daily 2-h extinction sessions as previously described (Feltenstein et al. 2007a; Fuchs et al.
2004b). Once extinction criterion was reached (defined here as a minimum of seven
extinction sessions with ≤15 active lever responses per session for the last two consecutive
days before testing), each rat underwent six separate reinstatement tests. Prior studies have
successfully utilized similar repeated reinstatement testing designs (Feltenstein and See
2006; Kippin et al. 2006). Rats experienced six total reinstatement tests examining three
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reinstatement factors before and after intracranial vehicle and B/M infusions. The following
three reinstatement triggers were used: conditioned cues, yohimbine administration (IP), and
conditioned cues+yohimbine. Each test was given twice with either vehicle or baclofen–
muscimol (B/M) infusions into the BNST immediately prior to the test. Both drug treatment
(vehicle or B/M) and reinstatement test type were counterbalanced. Animals that did not
finish all six reinstatement tests (e.g., due to cannulae blockade) were not utilized for data
analysis. Animals were extinguished to criterion between reinstatement tests (≤15 active
lever responses per session for two consecutive days). Yohimbine injections (2.5 mg/kg, IP)
were given 30 min prior to testing, and saline vehicle was given prior to conditioned cue
reinstatement tests. The yohimbine dose was based on previous reinstatement studies in rats
(Feltenstein and See 2006; Shepard et al. 2004). Conditioned cue-induced reinstatement tests
involved contingent presentation of the light+tone stimulus previously associated with the
active lever press during self-administration. Cue presentation was followed by a 20-s time-
out, during which lever presses were recorded, but had no programmed consequences.
Intracranial infusions
For intracranial infusions, stainless steel injection cannulae (33 gauge, Plastics One) were
inserted to a depth of 2 mm below the tip of the guide cannulae immediately prior to
placement into the chamber. The injection cannulae were connected to 10-ml Hamilton
syringes (Hamilton Co., Reno, NV, USA) mounted on an infusion pump (Harvard
Apparatus, South Natick, MA, USA). A combination of B/M (1.0/0.1 mM, 0.2 ul volume) or
phosphate-buffered saline vehicle (pH=7.0 for both solutions) was infused bilaterally over a
2-min time period. Dose–response analyses have shown that this concentration of B/M site-
selectively attenuates cocaine-primed (McFarland and Kalivas 2001) or conditioned cue-
induced reinstatement (Fuchs et al. 2004b) of cocaine seeking, and this dose of B/M has
previously been used to selectively inactivate the BNST (Rogers et al. 2008). The injection
cannulae were left in place for 1 min prior to and after the infusion.
To assess the effects of B/M inactivation on general motor activity, subsequent to
completion of reinstatement testing, a subset of animals were tested for locomotor activity
after vehicle or B/M infusions (n=10/group) into the BNST. Infusions occurred immediately
before placement into clear Plexiglas chambers (22×43×33 cm). Each chamber was
equipped with a Digiscan monitor (Omnitech Electronics, Columbus, OH, USA) containing
a series of 16 photobeams (eight on each horizontal axis) that tabulated total distance (cm)
traveled by each animal. Beam breaks were detected by a Digiscan analyzer and recorded by
DigiPro software (Verson 1.4). Immediately following a 1-h testing period, animals were
returned to their home cages.
Histology and data analysis
After completion of reinstatement testing, the rats were deeply anesthetized with equithesin
and transcardially perfused with PBS and 10% formaldehyde solution and processed as
previously described (Fuchs et al. 2004b). The most ventral point of the microinjector tips
were mapped onto schematics from a rat brain atlas (Paxinos and Watson 1997).
Reinstatement of responding from extinction levels and the effects of B/M inhibition of the
BNST on reinstatement were analyzed using separate one-way analysis of variance
(ANOVA) for each reinstatement condition, followed by pairwise comparisons with the
Student–Newman–Keuls test. Locomotor activity was measured in 12 5 min time bins and
analyzed with a two-way ANOVA (group×time). Data points were not included if they were
three standard deviations beyond the group mean. Analyses were considered statistically
significant at p<0.05. All data are presented as mean±SEM.
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Results
Histology
Inspection of injection cannulae tip locations showed that all correct BNST placements were
within the dorsomedial portions of the more posterior BNST, including the anterolateral and
anteromedial nuclei (Fig. 1a). A representative photomicrograph is shown in Fig. 1b. Note
that due to overlap, some points indicate injector locations for multiple animals. A total of
n=18 animals had correct injector placements in the BNST. Animals (n=8) with inaccurate
cannulae placements served as anatomical controls to validate the specificity of BNST
inactivation on reinstatement. These placements targeted the anterior commissure at various
rostral/caudal levels, as well as the internal capsule and the nucleus of the commissural stria
terminalis. It is important to note that the animals with placement outside the BNST did not
show any effect of B/M treatment on reinstatement responding (shown below).
Cocaine self-administration and extinction
Rats readily acquired cocaine self-administration and demonstrated stable patterns of active
lever responding and cocaine intake throughout the maintenance phase of the experiment.
Figure 2a indicates the mean lever responding for the last 2 days of cocaine self-
administration before extinction and reinstatement testing (mean active lever
responses=54.5±14.3 and mean daily cocaine intake= 20.7±2.3 mg/kg). Furthermore, all
animals decreased responding during extinction sessions in the absence of cocaine infusions
and cue presentations (Fig. 2b). Active lever responding reached the established extinction
criterion (<15 active lever responses over 2 days) in a mean of 8.7± 0.4 days before
subsequent reinstatement testing. Mean lever presses before the first reinstatement test was
measured across animals (mean active lever responses= 9.5±1.1). Inactive lever responding
showed uniformly very low levels throughout the study, and no significant differences were
found between treatment conditions during reinstatement trials (data not shown).
Spontaneous locomotor activity measured after intracranial infusions of either vehicle or B/
M (n=10/group) showed no significant effects of BNST inactivation on total locomotor
activity (data not shown).
Reinstatement testing
Reinstatement data for animals with correct placements in the BNST are shown in Fig. 3.
Four out of 162 datapoints were excluded on the basis of outlier criterion (>3 SD).
Presentation of conditioned cues led to significant reinstatement of responding on the
previously cocaine-paired lever during testing (F2,51=18.28, p<0.0001). Post hoc analyses
revealed significant reinstatement over extinction levels (p<0.05) and blockade of
conditioned cue reinstatement by intracranial B/M administration (p<0.05). Yohimbine
administration alone led to modest, but significantly increased reinstatement responding
(F2,48=3.94, p<0.05) above that of extinction levels (p<0.05). Yohimbine-induced
reinstatement was significantly attenuated by BNST inactivation (p<0.05). As previously
reported (Feltenstein and See 2006), pretreatment with yohimbine increased cue-induced
reinstatement of cocaine seeking beyond that seen with either cues or yohimbine alone.
Analyses revealed a significant main effect (F2,50=17.32, p<0.0001), with pronounced
reinstatement over extinction levels (p<0.05) as well as inhibition of the cue+yohimbine-
induced reinstatement by B/M (p<0.05). In animals with cannulae placements outside the
BNST, infusion of B/M had no effect on reinstatement of cocaine seeking under any of the
three conditions (Fig. 4), with significant responding over extinction levels seen for cue-,
yohimbine-, and cue+ yohimbine-induced reinstatement after either vehicle or B/M
infusions (p<0.05).
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Discussion
The current study shows a role for the BNST in both conditioned cue and yohimbine-
induced reinstatement of cocaine seeking in an animal model of relapse. Further, our results
effectively demonstrate blockade of the interactive effects of yohimbine stress and cues on
reinstatement, supporting the role of the BNST as a critical modulator when both types of
reinstatement factors are applied. While the role of the BNST in footshock stress-induced
reinstatement has previously been established (Erb and Stewart 1999; McFarland et al.
2004), examination of the neural substrates of conditioned cue-induced reinstatement has
focused primarily on the basolateral and central regions of the amygdala, rather than the
extended amygdala (See 2005). In addition to a role for the BNST in conditioned cue-
induced reinstatement, our results indicate that the BNST is critical for the interaction of
yohimbine stress and cues in the promotion of cocaine seeking, as BNST inactivation
blocked this potent form of reinstatement. These results suggest that stress-cue interactions
may be the result of the convergent activation of two separate, yet integrated stress and cue
pathways that include the BNST.
Previous studies examining the neural circuitry underlying stress-induced reinstatement have
generally utilized intermittent footshock stress (Ahmed and Koob 1997; Erb et al. 1996; Erb
and Stewart 1999). Several other forms of stress (e.g., restraint stress) have been found to
not produce reinstatement of drug seeking (Shaham et al. 2000). To date, studies have not
directly examined the neural circuitry underlying yohimbine stress-induced reinstatement, or
the degree to which patterns of neural activation may be similar during footshock or
yohimbine stress-induced reinstatement. Interestingly, both of these stressors (but not
several other types of stressors) induced similar increases in c-fos mRNA in the nucleus
accumbens shell, and the basolateral and central amygdalar nuclei (Funk et al. 2006).
However, other data indicate that yohimbine may promote reinstatement via mechanisms
distinct from those underlying footshock stress-induced reinstatement. While footshock
stress-induced reinstatement is thought to rely on interactions of CRF and NE within the
BNST (Leri et al. 2002), recent studies suggest that yohimbine stress-induced reinstatement
is unaffected by CRF receptor antagonists, or by NE α-2 agonists (Brown et al. 2009).
However, we have recently found blockade of yohimbine-induced and conditioned cue-
induced reinstatement by systemic administration of guanfacine, an α-2 receptor agonist
(Buffalari and See 2009b). Furthermore, the current results suggest that yohimbine and
footshock do both rely on intact function of the bed nucleus of the stria terminalis.
While yohimbine has been well characterized as an enhancer of NE activity via NE α-2
antagonist effects, other mechanisms in both the BNST and other brain regions may
contribute to the effects of the drug, in particular the enhancing effect of yohimbine on cues.
Yohimbine does have some affinity at serotonin (Millan et al. 2000) and dopamine (Scatton
et al. 1980) receptor subtypes, any of which may contribute to reinstatement of drug seeking.
Alternatively, NE increases in other terminal regions may contribute to reinstatement by
both yohimbine stress alone and the enhanced reinstatement with simultaneous stress and
cues. Yohimbine-induced increases in NE release in the basolateral amygdala (Buffalari and
Grace 2009) may enhance the motivational salience of conditioned cues, while increased
prefrontal NE tone (Garcia et al. 2004) could alter attentional processes and further
modulate cue salience.
While yohimbine does not induce a stress state fully analogous to an external stress situation
(e.g., social stress), the use of yohimbine as a stressor for an animal model of relapse offers
several experimental and translational advantages. First, yohimbine has well-characterized
stress and anxiety effects in humans (Southwick et al. 1999) including increased anxiety and
activation of the hypothalamic–pituitary adrenal axis (Charney et al. 1987), elicitation of
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panic attacks, and increases in blood pressure (Vasa et al. 2009). Yohimbine causes stress
and anxiety responses in animals as well, including increased cortisol in rats (Banihashemi
and Rinaman 2006) and monkeys (Lee et al. 2004), and increased anxiety-like behaviors in
several paradigms (File 1986; Johnston and File 1989; Bijlsma et al. 2010). Yohimbine
therefore offers a homologous method of stress activation across species. Second, while not
yet systematically tested for cocaine, yohimbine increases drug craving in opioid-dependent
patients (Stine et al. 2002). Also, yohimbine reliably reinstates drug-seeking behavior in rats
(Banna et al. 2010; Feltenstein and See 2006; Le et al. 2005; Shepard et al. 2004) and
monkeys (Lee et al. 2004). Finally, yohimbine has a relatively long half-life of several hours
(Hubbard et al. 1988), which allows for maintained stress activation across the duration of a
reinstatement test session, with or without conditioned cue exposure. Further exploration of
both the neural circuitry and pharmacological features of yohimbine stress-induced
reinstatement offers a promising direction for future studies.
While previous studies have delineated various aspects of the neural circuitry underlying
conditioned cue-induced reinstatement (Feltenstein and See 2008; See 2005), further studies
will need to establish the links between amygdalar-based circuitry and the monoaminergic
modulation of the corticostriatal circuitry necessary for reinstatement of drug seeking. The
lack of a direct projection from the basolateral amygdala to the ventral tegmental area
(VTA) suggests that the amygdala may modulate dopaminergic output in the prefrontal
cortex via direct projections to the prefrontal cortex or indirect projections via the central
amygdala regions and the BNST (Alheid 2003), which have glutamatergic, GABAergic, and
peptidergic projections to dopamine neurons (Georges and Aston-Jones 2001; Morrell et al.
1984). Cue presentation may activate basolateral amygdala efferents to these regions,
resulting in enhanced dopamine release in the prefrontal cortex. The importance of cortical
dopamine function is evidenced from studies that have shown attenuation of both
conditioned cue- (Ciccocioppo et al. 2001; See 2009) and footshock stress-induced (Capriles
et al. 2003) reinstatement by dopamine receptor blockade. The BNST may modulate cortical
dopamine levels via its projections to the VTA (Georges and Aston-Jones 2001, 2002).
Multiple changes in the BNST have been characterized after acute or repeated
noncontingent cocaine, as well as cocaine self-administration (Koob 2003). Cocaine has
been reported to increase dopamine in the BNST (Carboni et al. 2000) and enhance BNST
excitatory transmission (Dumont et al. 2005). Dopamine has also been shown to enhance
glutamatergic transmission in the BNST and modulate short-term NMDA-dependent
plasticity (Kash et al. 2008). Plasticity within the neural circuitry associated with
reinstatement has been implicated as a crucial component of cocaine-induced behavioral
changes that may underlie relapse (Kauer and Malenka 2007). Further, chronic cocaine
disrupts plasticity within the BNST, and leads to changes in NE transporter binding. NE in
the BNST, which is critical for footshock-induced reinstatement (Leri et al. 2002), causes
complex inhibitory and excitatory effects that are receptor and subregion dependent (Egli et
al. 2005). Finally, the BNST has been heavily implicated in withdrawal from abused drugs
(Koob 2003; Smith and Aston-Jones 2008), and withdrawal also disrupts long-term
potentiation of intrinsic excitability within the BNST (Francesconi et al. 2009). Such studies
complement the current results and suggest that the BNST is a critical region that may
undergo changes relevant to chronic drug use, craving, withdrawal, and relapse.
The BNST is a complex brain region with as many as 12 anatomically identified subregions
(Dumont 2009). However, the functional dissociation of different regions of the BNST,
particularly with regard to reward and addiction-related functions, has yet to be delineated.
Our placements primarily targeted the dorsomedial subdivisions, primarily in the posterior
portions of BNST. However, this placement was not exclusive, and inactivation of other
areas of the BNST was also effective. This is not surprising, in that other studies examining
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the role of the BNST in drug withdrawal, reinstatement, and reward have identified a role
for multiple subregions of the BNST (Aston-Jones et al. 1999; Delfs et al. 2000; Harris and
Aston-Jones 2003; Jalabert et al. 2009; Leri et al. 2002). Most recently, strong interactions
between the prefrontal cortex, multiple BNST subregions, and dopamine neurons of the
VTA have been identified (Jalabert et al. 2009). These interactions are relevant to the
current results, as the VTA and prefrontal cortical regions have been shown to play a role in
other forms of reinstatement of drug seeking (McFarland et al. 2004; See 2005).
We chose B/M infusions as a means of inactivation for four primary reasons: (1) sparing of
effects on fibers of passage, (2) targeting of both GABA A and B receptors, (3) extensive
previous research with this approach in determining the neural circuitry of reinstatement
(McFarland et al. 2004; McFarland and Kalivas 2001), and (4) previous experience in our
laboratory using B/M to specifically inactivate the BNST. However, as with any
pharmacological approach, the use of B/M necessitates careful interpretation of the resultant
behavioral effects. Often characterized as consisting of GABAergic interneurons and
projection neurons, recent investigations (Jalabert et al. 2009) have suggested that this may
be an oversimplification of the BNST. While combined GABA A and B agonists generally
result in an overall inhibition, in a structure with many interconnected GABAergic
interneurons (such as the BNST), more complex interactions could arise. Follow-up studies
will need to target specific neurotransmitter receptors, with a primary focus on NE receptors
due to yohimbine’s actions on the NE system and prior evidence of a role for BNST NE in
footshock-induced reinstatement of drug seeking (Leri et al. 2002).
The current results have implications for the phenomena whereby heightened stress
increases the likelihood of relapse in drug-dependent individuals in isolation, or when they
experience stimuli or environments associated with prior drug use (Sinha et al. 2006).
Whereas previous clinical and preclinical studies have almost exclusively focused on the
isolated effects of stress or cues, users usually encounter multiple triggers for relapse during
abstinence, and are more at risk during periods of greater stress or other maladaptive states,
such as anxiety or depression (Brady et al. 2007). Thus, the use of animal models aimed at
understanding the neurobiology of relapse need to incorporate the interactions that occur
between factors that promote relapse. The discovery of novel neural substrates underlying
the interaction of stress and cues in reinstatement will ultimately facilitate the development
of more effective interventions that can successfully target both domains that contribute to
relapse.
Acknowledgments
This research was supported by the National Institute on Drug Abuse grants DA16511 and DA21690 (RES), 1F32
DA025411-01 (DMB), and NIH grant C06 RR015455. The authors thank Anthony Carnell, Alisha Henderson, and
Bernard Smalls for technical assistance and data collection.
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Fig. 1.
a Schematic diagram illustrating placements of injection cannulae as confirmed through
histology (modified from Paxinos and Watson 1997). Coronal sections depicted are −0.2 to
−0.6 mm from bregma along the A/P coordinates. Placements are shown within (circles) or
outside of (triangles) the BNST. Please note some overlap in placements. The majority of
BNST placements were in the dorsomedial portions of the BNST, with a few placed in more
ventral locations. b A representative photomicrograph of BNST placements
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Fig. 2.
Responses (mean±SEM) on the active (ACT) and inactive (INACT) levers during cocaine
self-administration (left panel) and the last 7 days of extinction responding before
reinstatement (right panel)
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Fig. 3.
Responses (mean±SEM) on the active (previously cocaine-paired) lever for the last day of
extinction responding before testing (EXT), and following saline vehicle or baclofen–
muscimol (B/M) infusions into the BNST for conditioned cue-induced reinstatement tests
(CUE), yohimbine-induced reinstatement tests (YOH), and yohimbine+cue-induced
reinstatement tests (CUE+YOH). Significant differences (Student–Newman–Keuls test) are
indicated for reinstatement compared with extinction level responding (*p<0.05) or vehicle
vs B/M (†p<0.05)
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Fig. 4.
Responses (mean±SEM) on the active (previously cocaine-paired) lever for the last day of
extinction responding before testing (EXT), and following saline vehicle or baclofen-
muscimol (B/M) infusions directed at the BNST for conditioned cue-induced reinstatement
tests (CUE), yohimbine-induced reinstatement tests (YOH), and yohimbine+cue-induced
reinstatement tests (CUE+YOH) in animals with cannulae located outside of the BNST.
Significant differences (Student–Newman–Keuls test) are indicated for reinstatement
compared with extinction level responding (*p<0.05)
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