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Glutamate receptor-dependent modulation of dopamine efflux in the nucleus accumbens by the basolateral,… Howland, John George 2001

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G L U T A M A T E RECEPTOR-DEPENDENT M O D U L A T I O N OF DOPAMINE E F F L U X IN THE N U C L E U S A C C U M B E N S B Y THE B A S O L A T E R A L , BUT NOT THE C E N T R A L , N U C L E U S OF THE A M Y G D A L A IN RATS. by JOHN GEORGE H O W L A N D B.A. (Hons.), The University of Saskatchewan, 1999 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF M A S T E R OF ARTS in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Psychology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH C O L U M B I A August 2001 © John George Howland, 2001 In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada Abstract Dopaminergic neurotransmission in the nucleus accumbens (NAc) has been implicated in reward-related behavior. Recent experiments have described distinct roles of the central (CeN) and basolateral (BLA) amygdala nuclei in the context of associative reward-related learning. Given their direct and indirect connections to the NAc and ventral tegmental area (VTA), both the B L A and CeN may interact with the mesoaccumbens D A system during associative learning. The present experiments were designed to test i f electrical stimulation of the B L A or CeN could increase dopamine (DA) efflux in the NAc. Microdialysis combined with HPLC-ED was used to monitor NAc D A efflux in freely moving rats. Results revealed that stimulation of the B L A (20 Hz, 10 sec, 300 uA) induced a long-lasting 25±4% increase in NAc D A efflux whereas CeN stimulation had no effect. Reverse dialysis of either the N M D A receptor antagonist A P - V (100 uM) or the AMPA/kainate receptor antagonist D N Q X (100 uM), but not the metabotropic glutamate receptor antagonist M C P G (100 uM), into the NAc blocked the stimulation-induced increase in D A efflux in the NAc. Intra-VTA infusion (2 uL, 0.5 uL/min, unilateral) of the reversible sodium channel blocker lidocaine (4%) significantly reduced basal D A levels for ~ 30 minutes but failed to suppress the increase in NAc D A efflux resulting from B L A stimulation. Consequently, we suggest that afferents from the B L A directly modulate D A efflux at the terminals in the NAc, not the cell bodies in the V T A . Results are discussed in the context of limbic-striatal interactions and associative learning. i i i T A B L E OF CONTENTS Abstract i i Table of Contents i i i List of Figures iv Acknowledgments v INTRODUCTION 1 METHODS 5 Subjects 5 Surgery 6 Microdialysis Procedure 6 Analysis of Dialysate Samples 7 Experimental Procedure 8 Pharmacological Experiments 9 Micro-infusion Experiments 9 Drug Preparation 10 Histology 11 Data Analysis 11 RESULTS 11 High frequency stimulation of the B L A , but not the CeN, induces a long-lasting increase in D A efflux in the NAc of freely moving rats 11 Increased N A c D A efflux following stimulation of the B L A is dependent on ionotropic glutamate receptors located in the NAc 14 Unilateral infusions of Lido into the V T A significantly reduce basal D A levels in the NAc but fail to attenuate the increased NAc D A efflux resulting from B L A stimulation 15 DISCUSSION 16 Evidence for presynaptic modulation of D A efflux in the NAc by the B L A 17 Stimulation of the CeN does not induce long-lasting changes in NAc D A efflux 21 Functional implications 23 REFERENCES 27 LIST OF FIGURES Figure 1. Different effects of high frequency stimulation of the B L A and CeN on D A efflux in the NAc. Figure 2. Reverse dialysis of iGluR antagonists into the NAc blocks the increase in NAc D A efflux observed following B L A stimulation. Figure 3. Effects of intra-VTA Lido infusion on NAc D A efflux evoked by stimulation of the B L A . Figure 4. Histology. A C K N O W L E D G M E N T S I would like to thank the following (in no particular order) for their contributions to the research discussed in this thesis: The Canadian Institutes of Health Research and the National Science and Engineering Research Council for financial support granted to my supervisor, Dr. Tony Phillips, and me. Fred Lepiane, Lucy Gregorios-Pippas, Christina Cheng, and Kitty So for their help with histology and various other technical aspects of this project. Pornnarin Taepavarapruk for spending a large amount of time teaching me the microdialysis technique. Without her help I doubt this project would have been nearly as successful as it turned out to be. Dr. Stan Floresco and Soyon Ann for helpful discussions relating to all aspects of this project. And finally, Dr. Tony Phillips for providing me with the knowledge, resources, and guided freedom to effectively complete this project. 1 Introduction A growing body of evidence suggests that appropriate behavioral responses to rewarding stimuli depend on the functional integrity of the nucleus accumbens (NAc). N A c neurons fire in response to reward (Apicella et al., 1991; Schultz et al., 1992; Shidara et al., 1998) whereas lesions of the NAc impair conditioned place preference by rats (Everitt et al., 1991). Given extensive inter-connections with limbic and cortical areas (Groenewegen et al., 1991; Sesack and Pickel, 1992) and its proposed role as a "limbic-motor" interface (Mogenson et al., 1980; 1993), the NAc may function in rewarding contexts by allowing reward-related information stored in limbic or cortical areas to influence on-going behavior (Everitt et al., 1999; Kalivas and Nakamura, 1999; Meredith and Totterdell, 1999). The acquisition of instrumental responding for food is impaired by injections of the N M D A receptor antagonist A P - V into the NAc core (Kelley et al., 1997), whereas lesions of the NAc core impair rats' performance on tasks relying on either Pavlovian associations or conditioned reinforcement (Everitt et al., 1999). Dopamine (DA) release in the NAc has been implicated in reward-related processing, although DA' s exact function continues to be debated (Ikemoto and Panksepp, 1999; Redgrave et al., 1999). The NAc receives a strong dopaminergic projection from the ventral tegmental area (VTA; Bjorklund and Lindvall, 1984; Groenewegen et al., 1991) and natural rewards such as food (Ann and Phillips, 1999) or access to a sexually receptive partner (Fiorino et al., 1997) increase D A efflux in the NAc. Rats self-administer D A receptor agonists directly into the NAc (Ikemoto et al., 1997), whereas intra-NAc injections of amphetamine (Colle and Wise, 1988) or D A antagonists (Stellar and Corbet, 1989) enhance or disrupt self-stimulation behavior, 2 respectively. Accurate responding in a conditioned reinforcement paradigm can also be enhanced by intra-accumbens infusions of amphetamine (Robbins, 1978; Robbins et al., 1983), an effect that is dependent on D A transmission in the NAc (Taylor and Robbins, 1986), in conjunction with neural activity in the central nucleus of the amygdala (CeN; Robledo et al., 1996) and the ventral subiculum of the hippocampus (vSub; Burns et al., 1993). The latter findings raise the possibility that limbic areas such as the amygdala and hippocampus may regulate DA efflux in the NAc and thereby influence reward-related learning. Reward-related learning is dependent on the amygdala (Robbins and Everitt, 1992; Davis, 1992; Baldwin et al., 2000). Recent studies using both aversive (Killcross et al., 1997) and appetitive (Hatfield et al., 1996; Parkinson et al., 2000) paradigms report distinct roles of the B L A and CeN in learning goal-directed responses. Generally, the integrity of the CeN is required when animals form Pavlovian-type associations between conditioned and unconditioned stimuli that alter behavior. In contrast, the B L A appears to play an essential role in learning new adaptive instrumental responses for reward. As mentioned, the potentiation of responding in a conditioned reinforcement paradigm by micro-infusions of amphetamine into the NAc shell is dependent on projections from the CeN to the NAc (Robledo et al., 1996). Others emphasize the importance of the projections from the B L A to the NAc in regulating N A c D A efflux during affective perception (Louilot and Besson, 2000) and stimulus-reward associations (Cador et al., 1989). Taken together, these data suggest that both the B L A and CeN may interact in different ways with the mesoaccumbens D A system to enable appropriate learning about rewarding stimuli. 3 Neuroanatomical data are consistent with a role for both amygdala nuclei in modulating D A levels in the NAc. The B L A sends extensive direct, compartmentalised, glutamatergic projections to the NAc (Groenewegen et al., 1991; McDonald; 1991b; Brog et al., 1993; Wright et al., 1996). Interestingly, glutamatergic projections from the B L A synapse on the same dendritic spines of medium spiny neurons as do tyrosine hydroxylase positive fibers from the V T A (Johnson et al., 1994). N M D A receptors have also been localized along the axonal processes of D A neurons in the N A c (Gracy and Pickel, 1996). Therefore, it is possible that afferent activity from the B L A interacts presynaptically with D A terminals in the NAc. The V T A also receives a small afferent projection from the rostral B L A (Phillipson, 1979) that could influence D A levels in the N A c by causing increased firing of the D A cell bodies in the V T A . Two recent studies examined the effects of stimulation of the B L A on D A levels in the NAc. Using in vivo chronamperometry in anaesthetised rats, Floresco et al. (1998) showed that brief high frequency stimulation of the B L A induced a complex pattern of changes in D A efflux in the NAc that included a sustained increase in D A efflux that lasted for « 30 minutes. Additional experiments revealed that this increase in D A efflux is mediated by ionotropic glutamate receptors (iGluR's) located within the N A c and did not depend on D A cell body firing in the V T A . Therefore, the B L A may regulate D A efflux in the N A c by a presynaptic mechanism. Glutamatergic afferents from the B L A to the medial prefrontal cortex (mPFC) also have been implicated in regulating interactions between the B L A and the mesoaccumbens D A system while the B L A is activated (Jackson and Moghaddam, 2001). As a result, the mPFC may regulate the access of 4 emotionally relevant information in the B L A to the motor effector sites via the N A c (Jackson and Moghaddam, 2001). The CeN may also interact with the mesoaccumbens D A system through several different pathways. The CeN projects directly to the V T A (Phillipson, 1979; Simon et al., 1979; Wallace et al., 1989; 1992) and sends a relatively minor projection directly to the NAc (Phillipson, 1979). The CeN also is well connected to areas such as the thalamus and lateral hypothalamus, both of which send afferents to the NAc ; therefore, the CeN may influence D A transmission in the NAc through poly-synaptic pathways (Brog et al., 1993; Zahm and Williams, 1997; Zahm, 1998; 2000). The present experiments were designed to test a number of specific hypotheses. Given the findings of Floresco et al. (1998), we postulated that high frequency stimulation of the B L A would induce a long-lasting significant increase in D A efflux in the NAc of freely moving rats. Electrical stimulation parameters were based on single unit recording studies demonstrating that neurons in the B L A can fire at frequencies ranging from 10 to 40 Hz when rats are presented with either naturally or conditionally rewarding stimuli (Muramoto et al., 1993; Uwano et al., 1995; Pratt and Mizumori, 1998). Additionally, we hypothesized that application of specific iGluR antagonists into the NAc would block the increased DA efflux observed following B L A stimulation. Given our hypothesis that these changes in D A efflux may occur due to a presynaptic mechanism operating on the D A terminals in the NAc, we also predicted that inactivation of the D A cell bodies in the V T A would not significantly affect the stimulation-evoked increase in N A c D A efflux, despite a significant reduction in basal D A efflux. Finally, a series of experiments tested whether high frequency stimulation of the CeN could increase D A efflux in the NAc of freely moving rats. Consideration of the data presented by Everitt and colleagues (Robledo et al. 1996; Everitt et al. 1999; Parkinson et al., 2000; Everitt et al. 2000) suggested that stimulation of the CeN would increase D A efflux in the NAc, most likely via increased firing of D A cell bodies in the V T A . D A efflux in the NAc was analyzed by a combination of in vivo microdialysis and high performance liquid chromatography with electrochemical detection (HPLC-ED) in freely moving rats in all experiments. This protocol offers several advantages over the anaesthetized preparation previously used (Floresco et al., 1998); most notably, the elimination of the uncontrollable effects of the anaesthetic on brain function. HPLC-ED also allows for the unambiguous, accurate measurement of D A levels. Finally, microdialysis allows drugs of interest to be locally administered to the brain by reverse dialysis through the probe. Reverse dialysis allows drugs to be discretely administered to particular brain sites for long periods of time with precise control over the dosage and location of administration. Methods Subjects: Male Long-Evans rats (Charles River Canada, St. Constant, Quebec, Canada) were used in all experiments. Rats were housed in individual plastic bins, in a temperature controlled colony room maintained at 25°C, with free access to food (Purina Rat Chow) and water. A l l rats were maintained on a 12-h light-dark cycle (lights on at 7:00 a.m.). Experiments were conducted in strict accordance with the standards of the 6 Canadian Council on Animal Care and were approved by the Committee on Animal Care at the University of British Columbia. Surgery: Surgery was performed on rats weighing 320-400 grams. A l l rats were anaesthetized with ketamine hydrochloride (100 mg/kg, i.p.; M T C Pharmaceuticals) and xylazine (10 mg/kg, i.p.; Rompun). They were then placed in a stereotaxic apparatus (Kopf, Germany), the dorsal skull surface was exposed, and holes were drilled. A bipolar stimulating electrode (Plastics One, Roanoke, V A ) was implanted into either the B L A (AP -3.4 mm from bregma, M L ±5.0 mm from the midline, D V - 7.6 mm from dura) or CeN (AP - 2.4 mm, M L ± 4.2 mm, D V -7.0 mm) and a guide cannula (19 gauge, 15 mm) was implanted dorsal to the ipsilateral NAc (AP +1.8 mm, M L ±1.1 mm, D V -1.0 mm). Rats used in the V T A lidocaine experiment also had a guide cannula (23 gauge, 20 mm) implanted dorsal to the ipsilateral V T A (AP - 4.8 mm, M L ±1.0 mm, D V - 6.5 mm). A l l coordinates were calculated using the rat brain atlas of Paxinos and Watson (1997). Cannulae and the electrode were secured to the skull with four jeweler's screws and dental acrylic. Wire obdurators were inserted into the cannulae to prevent foreign material from entering the brain during the recovery period. Rats were given at least 5 days to recover from surgery before testing. Microdialysis Procedure: Concentric-style microdialysis probes were constructed in our laboratory. They consisted of a 24-gauge stainless steel cannula (34 mm), fused silica tubing (102 urn i.d. x 165 um o.d., PolymicroTechnologies Inc, Phoenix, AZ) , polyethylene tubing (PE 50, Intramedic, Becton Dickinson, USA), and a semi-permeable hollow-fiber membrane (2.0 mm of exposed membrane, 340 urn o.d., 65 kDa molecular weight cutoff, Filtral 12, Hospal-Gambro, Germany). Quick drying epoxy glue (Lepage 12) was used to secure the joints of the probe and plug the tip of the dialysis membrane. The day before the experiment was conducted, probes were attached to gas-tight syringes (Hamilton, Reno, Nev.) via a liquid swivel (Instech, Inc., Plymouth Meeting, Pa.) containing perfusion medium (147 mM NaCl, 3.0 m M KC1, 1.3 m M CaCl 2 .H 2 0, 1.0 m M MgCl 2 .6H 2 0, 0.01 sodium phosphate buffer; pH 7.3-7.4). Probes were flushed at 2 uL/min for 10 to 20 minutes using a syringe pump (Model 22, Harvard Apparatus, South Natick, M A ) . Probes were then attached to a copper collar and inserted into the N A c (7.8 mm ventral to dura) through the guide cannula previously implanted dorsal to the NAc . At this time, the lead for the electrode was also secured to the electrode. Rats were tested two at a time in a room containing two small Plexiglas testing chambers (32 cm x 32 cm x 41 cm high). Once the probe was inserted and the electrode lead attached, the rat was allowed to move freely in the plexi-glass box with access to food and water for 12 to 18 hours before experimental testing began the following morning. Throughout the night, the microdialysis probes flowed continuously at 1 uL/min and a timer for the lights in the testing room ensured the light-dark cycle remained the same as in the colony room. Analysis of Dialysate Samples: Two HPLC-ED were used to quantify D A levels in all experiments. Each system consisted of an ESA 582 pump (ESA Inc., Bedford, M A ) , Rheodyne Inert manual injector (Rheodyne, Rohnert Park, CA) , an Ultrasphere column (Beckmann, Fullerton, CA. ; ODS 5 urn, 15 cm x 4.6 mm), an ESA 5011 analytical cell, and a Coulochem II EC detector (ESA Inc.). The working potentials were: +450 mV 8 (electrode 1), -300 mV (electrode 2), and +450 mV (guard cell). The mobile phase consisted of 6 g/L sodium acetate, 10 mg/L ethylenediaminetetra-acetic acid (EDTA), 150 mg/L octyl sulfate (adjustable), 35 ml/L glacial acetic acid and 865 mL Mi l l i Q purified water. The pH of the mobile phase was adjusted to 3.5 with glacial acetic acid, and it was filtered through a 0.22 urn sterile nylon filter unit (Millipore, Bedford, M A ) . Methanol (HPLC grade, 10 % of total volume, adjustable) was added after filtering and the mobile phase was degassed prior to use. Chromatograms were registered on a dual-pen chart recorder (Kipp and Zonen, Bohemia, NY) . A l l samples were injected into one of the HPLC systems immediately after collection and D A peak heights were manually measured. Experimental Procedure: A within-subjects design was used in all experiments and was combined with a between subjects design for the V T A micro-infusion experiments (see below). Baseline samples were collected every 10 min from the animals starting at 8-10 a.m., 14-18 hrs after the probes were inserted into the NAc. After four baseline samples were collected that did not differ by more than ± 1 0 %, the rats received electrical stimulation. Cathodal constant current pulses were delivered to either the B L A or CeN through an isolater (Iso-flex, A.M.P.I. , Israel) via a Master-8 stimulator (A.M.P.I.). Two hundred pulses were delivered at an intensity of 300 uA and a frequency of 20 Hz for 10 seconds. The intensity of stimulation was based on a similar protocol used for stimulation of the ventral hippocampus in our laboratory (see Taepavarapruk et al., 2000 for details). Stimulation was delivered at an appropriate time to ensure the next sample reflected only "stimulation-evoked" changes in D A efflux. The experimenter remained in the testing 9 room following the stimulation to record any behavioral effects of the stimulation. Following the first stimulation, dialysate samples were taken collected every 10 min and analyzed until further manipulations were performed (see below and results section for details). Pharmacological Experiments (performed only in conjunction with BLA stimulation): In separate groups of rats, reverse dialysis was used to deliver the a-amino-3-hydroxy-5-methyl-4-isoxazoleprionic acid (AMPA)/kainate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX), the N-methyl-D-aspartate (NMDA) receptor antagonist (±)-2-amino-5-phosphonopentanoic acid (AP-V), or the broad spectrum metabotropic glutamate receptor antagonist (+)-a-methyl-4-carboxyphenylglycine (MCPG) to the NAc through the same probe as was used to gather DA. The dose in the perfusion medium for all drugs was 100 uM. Previous experiments (Taepavarapruk et al., 2000; Taepavarapruk and Phillips, unpublished observations) indicated that an intra-probe dose of 100 u M is sufficient to effectively block glutamatergic receptor activation in the NAc with these receptor antagonists. Perfusion of the drug into the N A c was started 20 min before the second stimulation of the B L A and continued for a total of 60 min. Micro-infusion Experiments (performed only in conjunction with BLA stimulation): Injection needles were constructed from 30-gauge stainless steel tubing and PE-50 tubing. The needles were filled with 4% lidocaine (Lido) before being inserted into the guide cannula implanted dorsal to the V T A . A short piece of PE-50 tubing secured the needles within the 23-gauge guide cannulae. A l l needles were inserted 1 mm past the end of the cannula into the V T A at least 2 hr before baseline sample collection began. The micro-infusion needles remained in the rats' brains throughout the experiment. Except during the micro-infusion procedure, the exposed end of the PE-50 tubing was plugged to ensure that Lido did not leak into the brain. Shortly before the injection began, the needles were connected to gas-tight syringes (500 uL, Hamilton, Reno, Nev.). The desired volume of Lido (VTA: 2 uL, ipsilateral to the electrode) was infused (0.5 uL/min) via syringe pump (Model 22, Harvard Apparatus) 10 min before the second stimulation. The experimenter confirmed the progress of the infusion by measuring the movement of an air bubble in the PE-50 line during the infusion. Five minutes after the infusion ended, rats in the Stim and StimCtrl groups were stimulated a second time while rats in the Ctrl group were not (see results section). Previous studies have shown that Lido's strongest effect begins ~ 5 min after its infusion (Tehovnik and Sommer, 1997). Dialysate samples were collected for 90 min following stimulation two in all experiments. Drug Preparation: Appropriate amounts of A P - V , DNQX, and M C P G (Precision Biochemical, Inc.; Vancouver, Canada) were dissolved in a drop of NaOH and water to create a 10 m M stock solution (volume was adjusted to 1 ml with perfusion medium; pH was adjusted to ~ 7.0). The stock solutions were then diluted to 100 u M with perfusion medium and stored at - 20 °C until use. Lido (4 %) was freshly made when needed by dissolving 20 mg/ml of lidocaine HC1 powder (RBI, Natick, M A ) into an injectible 2 % lidocaine solution (Ayerst, Guelph, Ont, Canada). Histology: Following the experiment, all rats were injected with a lethal dose of chloral hydrate and perfused transcardially with 0.9 % saline followed by 10 % formaldehyde. Brains were stored in 10 % sucrose in 10 % formaldehyde for at least 1 week, after which they were sectioned (50 um) using a cryostat and stained for Nissl substance with cresyl violet. Placements of the probes, electrodes and infusion needles were verified under a light microscope with the assistance of a rat brain atlas (Paxinos and Watson, 1997). Data Analysis: D A peak heights were measured on the chromatograms and were entered into a Microsoft Excel spreadsheet. In all figures, D A levels are represented as a percentage of the first three baseline samples included in the analysis. The baseline sample taken immediately before stimulation was used as the critical value during computation of the Dunnett's post-hoc tests. Within-subjects analysis of variance were performed with the aid of SPSS (version 10.0). Dunnett's post-hoc tests were used where appropriate. To assess differences between groups in the VTA-Lido study, multiple t-tests were performed (a levels adjusted accordingly). Results High frequency stimulation of the BLA, but not the CeN, induces a long-lasting increase in DA efflux in the NAc of freely moving rats. During the baseline sampling periods, rats were typically sleeping or resting in the testing chambers. As shown in Fig. IA, baseline D A levels were very stable. High frequency stimulation (20 Hz, 10 sec, 300 uA; n=7) of the B L A induced a significant (F24, H4=3.15, p<0.0001 and Dunnett's, p<0.05) increase in D A efflux in the N A c that 12 returned to baseline 30 min after the stimulation (Fig. \A). A l l rats received a second train of brain-stimulation 2 hr (12 samples) after the first stimulation. This stimulation also produced a significant increase in D A efflux in the NAc very similar to that observed after the first stimulation (Dunnett's, p<0.05). DA levels returned to baseline 40 min after stimulation, and the experiment was ended 90 min after the second stimulation. Throughout testing, rats generally slept/rested except for exhibiting a number of characteristic behavioral responses immediately following stimulation of the B L A . A l l rats used in the B L A stimulation experiments (37/37) woke up during the first 2 to 4 seconds of stimulation and most stood up during this time. Fifty-two percent of rats exhibited significant chewing for less than 10 sec after the stimulation began, an additional 30 % exhibited chewing that lasted for more than 10 sec, and 18 % showed no visible signs of chewing during the 5 min following the start of stimulation. A characteristic rapid turn between 90° and 270° during the stimulation was displayed by 35 % of rats. In 87 % of these rats, the turn was ipsilateral to the electrode. Twenty-five percent of the rats returned to a sleeping/resting position less than 2 min after brain stimulation, while an additional 64 % returned to sleeping/resting 2 to 5 min after the stimulation. The remainder of the rats (11 %) were active for longer than 5 min after the stimulation. Some of the rats (16 %) displayed at least one wet dog shake after the stimulation. The behavioral responses exhibited by a given rat were similar after the first and second stimulation. A significant stimulation-evoked increase in D A efflux in the NAc was observed only when electrode placements were located in the caudal B L A (n=37; Fig. 4A). The majority of effective placements (shown on the left side of the sections) were centered in 13 the caudal magnocellular accessory basal and parvicellular basal nuclei (-3.3 mm, -3.6 mm, and -3.8 mm from bregma). The right side of the sections depicts a representative sample of placements from rats which, when stimulated, did not show an increase in D A efflux in the NAc (n=31). These placements were located in the lateral nucleus of the amygdala, ventral piriform cortex, CeN, bed nucleus of the stria terminalis, and caudate-putamen. Some rats represented by placements shown on the right side of this figure were used in the control groups for the VTA-Lido experiments discussed below. Representative placements of the microdialysis probes used in all experiments are seen in Fig. AC. Many of the probes were centered on the border between the shell and core of the NAc, although some sampled primarily from either the shell or core components. In contrast to the data obtained for the B L A , electrical stimulation of the CeN (20 Hz, 10 sec, 300 uA; n = 6) failed to produce a significant change in D A efflux in the N A c (data not shown; F9; 45=1.32, n.s.). To ensure that this finding was not due to insufficient current intensity, 8 naive animals were implanted with electrodes in the CeN and stimulated at intensities of 300, 450, 600, and 800 uA with 60 min between each stimulation. Dialysis samples were collected continuously for the duration of this experiment, and no significant change in NAc D A efflux was detected at any of these current intensities (Fig. 15, F27,189=1.34, n.s.). Behavioral effects of CeN stimulation were similar to those of B L A stimulation (64 % of CeN-stimulated rats exhibited chewing behavior). Those rats in the parametric study (300-800 uA) displayed an increase in the duration of chewing behaviors at higher current intensities. Stimulating electrodes in the CeN group were located in the lateral and medial divisions of the CeN (n=14; Fig. 45). 14 Increased NAc DA efflux following stimulation of the BLA is dependent on ionotropic glutamate receptors located in the NAc. Reverse dialysis experiments examined the role of both ionotropic and metabotropic glutamate receptors in the NAc on mediating the effects of B L A stimulation on NAc D A efflux. Control stimulation of the B L A in the groups given the iGluR antagonists A P - V (n=7) or D N Q X (n=8) resulted in a significant increase in D A efflux in the NAc similar to that observed in the initial stimulation experiment (AP-V group, F24. 144= 2.34, p<0.001, and Dunnett's, p<0.05; D N Q X group, F24,168= 4.23, pO.OOOl, and Dunnett's, p<0.05). Reverse dialysis of either A P - V (100 uM) or D N Q X (100 uM) for 20 min before, and 40 min after, the second B L A stimulation blocked the increase in D A efflux (Fig. 2A and B, Dunnett's, n.s.). Reverse dialysis of the metabotropic glutamate receptor antagonist M C P G (lOOuM) failed to block the stimulation-induced increase observed in a separate group of rats (n=6, F 24 ; 120= 1-72, p<0.05, Dunnett's, p<0.05 for both the first and second stimulation). Application of these drugs did not change the behavior of the rats or significantly affect basal D A levels. However, as can been seen in Fig. 25, reverse dialysis of D N Q X into the NAc did cause a small, non-significant (10±6%) decrease in basal D A levels in the N A c 10 min after its application began (immediately preceding stimulation 2). This effect has been seen previously (Taepavarapruk et al., 2000) and may reflect a role for AMPA/kainate receptors located in the NAc in regulating basal levels of D A release. 15 Unilateral infusions of Lido into the VTA significantly reduce basal DA levels in the NAc but fail to attenuate the increased NAc DA efflux resulting from BLA stimulation. This experiment assessed the contribution of D A cell body firing in the V T A to the increase in NAc D A efflux following stimulation of the B L A . The general design was the same as the previous experiments and three groups of rats were used. Following baseline sampling, the group receiving B L A stimulation (Stim group, n = 9) showed the characteristic, significant increase in NAc D A efflux (F21,168= 7.24, p<0.0001, Dunnett's test, p<0.05) as observed in the previous experiments. In control rats, neither the "Ctrl" (n = 7) nor the "StimCtrl" (n = 5) groups showed a significant increase in N A c D A efflux in response to brain stimulation (Dunnett's test, n.s.). As shown in Fig 4B, stimulation of only the caudal B L A resulted in a significant increase in D A efflux in the NAc. Histological analysis revealed that the stimulating electrodes in the Ctrl and StimCtrl groups were positioned outside this area. Micro-infusion of Lido into the V T A (2 uL, 0.5 uL/min, ipsilateral to the electrode) significantly reduced basal D A levels in all three groups (Stim group, Dunnett's, p<0.05; Ctrl group, F21,126= 4.76, Dunnett's, p<0.05; StimCtrl group, F21,84 =3.88, Dunnett's, p<0.05) consistent with an effect of Lido in reducing D A cell body firing in the V T A . As can be seen from Fig. 3 A, D A levels in the three groups were reduced by 35 to 40 % 10 min after infusion of Lido (no significant differences between groups, p>0.05). D A levels in the Ctrl group remained significantly below baseline for the next 20 min (Dunnett's, p<0.05) reaching a nadir of 54 ± 4% below baseline 20 min after the infusion of Lido was terminated. As expected, stimulation of rats in the 16 StimCtrl group following the Lido infusion did not change D A levels relative to the Ctrl group, and similar to the Ctrl group, levels of D A efflux remained significantly below baseline values for a total of 30 min (Dunnett's, p<0.05). In dramatic contrast to these data, stimulation of the B L A of the rats in the Stim group caused a significant increase in N A c D A efflux (Dunnett's, p<0.05). Although D A levels in the Stim group did not increase above baseline (due to the ongoing reduction of D A cell body firing in the V T A due to the infusion of Lido), their D A levels were 42 % higher than those of the two control groups (Fig. 35). D A levels in the Stim group remained significantly higher than those of the two control groups for at least 10 min (Stim vs. Ctrl, ti4=6.64, p<0.01; Stim vs. StimCtrl, ti4=3.68, pO.Ol;) after which D A levels in all three groups returned to baseline levels. Placements of the micro-infusion needles aimed at the V T A for the animals used in this experiment are shown in Fig. 4D. Discussion The main results of these experiments can be summarised as follows. High frequency stimulation of the B L A induced a significant long-lasting increase in D A efflux in the NAc, whereas stimulation of the CeN at four current intensities failed to change D A efflux significantly in the NAc. Reverse dialysis of the N M D A and AMPA/kainate receptor antagonists A P - V and D N Q X into the NAc effectively blocked the B L A stimulation-induced increase in NAc DA efflux, whereas reverse dialysis of the mGluR antagonist M C P G had no effect. Additional experiments revealed that micro-infusions of Lido into the V T A did not affect on the stimulation-evoked increase in D A 17 efflux in the NAc. Taken together, these data suggest that changes in neuronal activity in the caudal B L A can evoke increases in D A efflux in the NAc via a direct glutamatergic afferent projection. Evidence for presynaptic modulation of DA efflux in the NAc by the BLA. Evidence from this study and others (Floresco et al., 1998) is consistent with the hypothesis that stimulation of the B L A increases D A efflux in the NAc via the direct glutamatergic pathway connecting these two brain regions. Histological evidence for effective brain-stimulation loci (Fig. AA) shows that only placements in caudal areas of the B L A induce changes in D A efflux in the NAc. These data are consistent with neuroanatomical studies (McDonald, 1991b; Brog et al., 1993; Wright et al., 1996) identifying a strong glutamatergic projection from the caudal B L A to the NAc . These effective placements are not located in close proximity to the origin of a minor projection to the V T A originating in the rostral B L A (Phillipson, 1979). Therefore, it is unlikely that BLA-evoked efflux of D A in the NAc is mediated by direct activation of neurons in the V T A . Glutamatergic and dopaminergic afferents overlap anatomically and functionally in the NAc. For example, glutamatergic B L A afferents synapse in close apposition to tyrosine hydroxylase containing varicosities on spines of individual medium spiny neurons (Johnson et al., 1994). In situ hybridization and immunohistochemical methods reveal that iGluR's are expressed in midbrain D A neurons (Sato et al., 1993; Standaert et al., 1994). More importantly, N M D A receptors are located on tyrosine hydroxylase containing varicosities in the NAc shell (Gracy and Pickel, 1996), thus providing a mechanism for presynaptic modulation of D A efflux by glutamate released from B L A afferents. Recent studies support a close interaction between iGluR activation and D A release in the NAc. The injection of glutamate or iGluR agonists into the NAc increases N A c D A efflux (Imperato et al., 1990; Desce et al., 1992; Ruzicka and Jhamadas, 1993; Youngren et al., 1993), and 6-OHDA lesions of the NAc reduce glutamate receptor binding in the NAc (French et al., 1985; Zavitsanou et al., 1996). Taken together, these findings provide strong support for a role of iGluR's in the presynaptic modulation of N A c D A efflux. It is also possible that increased glutamatergic transmission in the NAc following B L A stimulation could activate mGluR's. mGluR binding sites are present in the NAc and V T A (Albin et al., 1991) and mGluR's appear to be formed in D A cell bodies located in the V T A (Testa et al., 1994). mGluRs are coupled to G-proteins and exert a variety of effects on ion channels and receptors located both pre- and post-synaptically (Pin and Bockaert, 1995), and have been hypothesized to reduce glutamate and/or D A release in the N A c (Floresco et al., 1998). In the present study, reverse dialysis of the broad-spectrum mGluR antagonist M C P G did not block the increase in NAc D A efflux following B L A stimulation, in contrast to the iGluR antagonists A P - V and DNQX. This finding is consistent with data from the anaesthetized preparation (Floresco et al., 1998), and suggests that the mechanism by which B L A stimulation increases N A c D A efflux is dependent selectively on activation of iGluRs. Results from the VTA-Lido experiment are consistent with the hypothesis that a presynaptic mechanism may underlie the B L A stimulation-induced efflux of D A in the NAc. These data confirm that stimulation of the caudal B L A can increase NAc D A 19 efflux even when D A cell body firing in the V T A is significantly reduced (Stim group; Fig. 3,4) whereas stimulation of areas adjacent to the caudal B L A (i.e. the lateral nucleus of the amygdala, ventral piriform cortex, CeN, bed nucleus of the stria terminalis, and caudate-putamen) has no effect on NAc D A efflux (StimCtrl group; Fig. 3A). Lido is a short acting (-30 minutes) reversible sodium channel blocker (Tehovnik and Sommer, 1997). Its effectiveness in inactivating a significant portion of the D A cell bodies in the V T A is evidenced by a significant 54 % reduction in D A levels in the control groups that lasted 30 min. Additionally, the return of D A efflux to pre-Lido baseline values in all three groups provides evidence that the infusion did not damage the mesoaccumbens D A system. These findings, in conjunction with the data obtained from reverse dialysis of iGluR antagonists into the NAc, convincingly demonstrate that stimulation of the B L A has a direct modulatory effect on D A terminals in the NAc. As mentioned briefly in the introduction, the mPFC may also play a role in mediating interactions between the B L A and mesoaccumbens D A system. The B L A sends a strong glutamatergic projection to the mPFC (Kelley et al., 1982; Groenewegen and Berendse, 1990; McDonald, 1991a) and the mPFC has reciprocal connections with the B L A and also sends afferents to both the NAc and V T A (Sesack et al., 1989; Sesack and Pickel, 1992; Berendse et al., 1992). Jackson and Moghaddam (2001) stimulated the B L A at low intensities for 10 min while monitoring D A efflux in the NAc and observed that D A efflux in the NAc was held constant by a glutamatergic dependent mechanism in the mPFC. Consistent with our data, once stimulation of the B L A was terminated, there was a long-lasting increase in D A efflux in the NAc (Jackson and Moghaddam, 2001). 20 Thus, these results raise the further possibility that activity in the B L A may influence D A efflux in the NAc through a poly-synaptic loop that includes the mPFC. An important difference between our protocol and that used by Jackson and Moghaddam (2001) is their continued application of low intensity bursts of stimulation (burst = 5 pulses in 20 msec, 1 sec inter-burst interval, intensity = 50 uA) to the B L A for 10 min in contrast to our delivery of 200 pulses of 20 Hz stimulation over a 10 sec period. Due to the brevity of our stimulation, it is not possible to measure D A efflux in the N A c during the stimulation with microdialysis. However, a short duration (~ 30 to 60 sec) increase in D A efflux time-locked to B L A stimulation has been observed using in vivo chronamperometry (Floresco et al., 1998). One strategy to determine whether or not the mPFC is involved in regulating the increase in D A efflux seen after B L A stimulation would be to inactivate the mPFC temporarily during B L A stimulation. If this manipulation fails to block the stimulation-evoked release of D A in the NAc , it could be inferred that a poly-synaptic projection from the BLA-mPFC-NAc does not modulate D A efflux in the NAc. This experiment is currently underway in our laboratory. If our prediction is validated, it will further strengthen our assertion that glutamatergic afferents from the B L A to the NAc modulate NAc D A efflux via a presynaptic mechanism. Several different mechanisms may explain the long-lasting changes in NAc D A efflux resulting from brief excitatory input from the B L A . It is clear, given our reverse dialysis data, that activation of both AMPA/kainate and N M D A receptors in the N A c is necessary for the stimulation-induced increase in D A efflux. Therefore, a C a 2 + -dependent form of potentiation may increase the efficacy of D A release in the D A cells. It is well established that activation of AMPA/kainate receptors by glutamate is sufficient 21 to relieve the Mg block on N M D A receptors that exists at resting membrane potential and allow them to be activated. Increased levels of intracellular C a 2 + resulting from N M D A receptor activation have been implicated in some forms of LTP (Bliss and Collingridge, 1993), probably as a result of increased Ca2+-dependent kinase activity. Increased activity of kinases such as calmodulin-dependent kinase II could increase D A efflux in the N A c by phosphorolating a number of potential sites, such as ionotropic receptors or presynatic vesicle docking proteins that make up the SNARE complex. Secondly, stimulation of the B L A may increase neuropeptidergic transmission in the NAc. Peptides such as cholecystokinin (CCK) and neurotensin are co-localised in projections from both the B L A and V T A to the N A c (Zaborszky et al., 1985; Seroogy et al., 1988), thus stimulation of the B L A could potentially increase their release in the NAc. Experiments performed both in vitro and in vivo have shown that C C K increases D A (Ruggeri et al., 1987; Marshall et al., 1991) and glutamate efflux (You et al., 1996) in the NAc. Therefore, an interaction between iGluR's and neuropeptide levels in the N A c may underlie the changes in D A efflux observed following B L A stimulation. Stimulation of the CeN does not induce long-lasting changes in NAc DA efflux. Surprisingly, stimulation of the CeN did not change D A efflux in the NAc. Neuroanatomical and excitotoxic lesion data in combination with a number of associative learning paradigms have led to the suggestion that the CeN interacts with the mesoaccumbens D A system at the level the V T A (Everitt et al., 1999; 2000). The CeN sends a GABAergic projection to the V T A (Wallace et al., 1992), but there is no direct evidence that it interacts with midbrain D A neurons in the rat. Recent evidence in non-22 human primates shows that CeN afferents to the midbrain do terminate in areas where tyrosine hydroxylase labelled cells exist (Fudge and Haber, 2000), although direct synaptic contact between the two cell types was not confirmed. Other researchers have emphasized the minimal nature of the projection from the CeN to the V T A (Zahm and Williams, 1997; Zahm, 1998) and suggested that the CeN may exert its strongest influence on the N A c indirectly through connections to the paraventricular nucleus of the thalamus and the caudal half of the lateral hypothalamus (Zahm, 1998; 2000). Activation of the CeN may produce short-lasting phasic changes in D A efflux in the NAc through either direct or indirect contacts with D A neurons in the V T A that are too brief to be detected by microdialysis samples of 10 min duration. Recent research from our laboratory (Ann and Phillips, 2001) has shown that inactivation of the CeN by reverse dialysis of Lido is accompanied by a significant decrease in N A c D A efflux. Given the extensive GABAergic interneuron network that exists in the V T A (Wallace et al., 1992), GABAergic afferents from the CeN may contact these cells and provide a degree of tonic inhibition during normal functioning. Removal of this inhibitory influence by Lido inactivation of the CeN could result in increased activity of interneurons in the V T A which in turn would suppress D A cell body firing in the V T A , thereby inhibiting D A efflux in the NAc (Ann and Phillips, 2001). If such a mechanism were involved, electrical stimulation of GABAergic afferents of the CeN could decrease the inhibitory tone of the GABAergic interneuron network within the V T A , thereby increasing the firing rate of DA neurons. This hypothesis will be tested by fast sampling of D A efflux in the NAc with in vivo chronoamperometry. 23 Functional Implications. Recently, Swanson and Petrovich (1998) proposed a theory of amygdala function based on the principle neurotransmitter species present in the efferent projections of the major nuclei in this region of the brain. Immunohistochemistry and in situ hybridization studies show a dense band of GABAergic cells extending from the caudoputamen, through the CeN and medial nucleus of the amygdala to the edge of the cerebral hemisphere, consistent with the assertion that the projections of the CeN are primarily GABAergic (Swanson and Petrovich, 1998). Other amygdala nuclei, such as the B L A , contain few GABAergic cells and have primarily glutamatergic efferent projections (Swanson and Petrovich, 1998). As the projections of the striatum and cortex are also primarily GABAergic and glutamatergic, respectively, these authors suggest that the CeN is a specialised striatal projection area for "regulating autonomic motor outflow" (p. 329) and that the B L A is a specialised cortical projection area that sends efferents to areas such as the NAc , caudoputamen, and prefrontal cortex (Groenewegen et al., 1991; Swanson and Petrovich, 1998). In light of these anatomical considerations, several specific predictions about the regulation of the mesoaccumbens D A system by each sub-region of the amygdala can be made. Given the sophisticated role of the cortex in the planning and execution of adaptive behavioral responses, it is postulated that the B L A exerts a selective and specialised modulation of the D A system. In contrast, the striatal-associated CeN may influence the D A system in a less specific, more global manner, consistent with its generalised effects on the autonomic and endocrine systems during stressful situations. 24 In the present study, stimulation of the B L A exerted a long-lasting influence on D A efflux in the NAc through a putative presynaptic mechanism. Recent experiments from our laboratory have demonstrated that high frequency stimulation of the B L A increases D A efflux in the NAc, and which in turn potentiates the ability of low frequency B L A inputs to the NAc to evoke firing in the medium spiny neurons (Floresco et al., 2001a). On the basis of these data, it is hypothesised that high frequency (>20Hz) activity in the glutamatergic afferents from the B L A can autoregulate and amplify the response of medium spiny neurons in the NAc to subsequent inputs across this pathway. Studies examining another cortical-type projection area of the limbic system, the ventral subiculum (vSub) of the hippocampus, have revealed a similar mechanism (Blaha et al., 1997; Taepavarapruk et al., 2000; Floresco et al, 2001b). High frequency stimulation of the vSub induced an increase in D A efflux in the NAc (Blaha et al., 1997; Taepavarapruk et al., 2000), which potentiated hippocampally-evoked firing of the medium spiny neurons in the N A c (Floresco et al., 2001b). Thus, overlapping cortical-type limbic projection areas can subtly affect the output of the NAc , primarily through potentiation of their own inputs to the NAc by direct regulation of the mesoaccumbens D A system. Data from the present experiments show that stimulation of the CeN does not have a long-lasting effect on NAc D A efflux, although its role in the tonic regulation of D A efflux in the NAc by effects on the DA cell bodies in the V T A remains a definite possibility (Ahn and Phillips, 2001). The regulation of D A efflux by connections to the D A cell bodies may lack the selectivity and behavioral specificity that the presynaptic modulation of specific D A terminals in the NAc by areas such as the B L A and vSub exhibits. However, a more general regulatory role of the CeN in D A modulation is 25 consistent with its proposed function in the activation of the autonomic and endocrine systems in response to stress (Davis, 1992; Swanson and Petrovich, 1998). A similar dissociation between the function of the CeN and B L A is supported by behavioral experiments designed to clarify the roles of the CeN and B L A in associative learning. Data obtained from rats tested in a concurrent conditioned-suppression and conditioned-punishment task showed that lesions of the CeN disrupt Pavlovian-type conditioned-suppression responses to the presentation of an aversive CS+, but spare the avoidance of future punishment by biasing responses away from an operant response related to the aversive CS+ (Killcross et al., 1997). In contrast, rats with lesions of the B L A suppressed their operant behavior immediately following the presentation of the CS+, but were impaired in their ability to inhibit responses that led to further presentations of the aversive CS+. Clearly, planning future actions to avoid punishment is a complex behavior that relies on the executive functions commonly attributed to the cortex, whereas Pavlovian-type behavioral reactions are often reflexive in nature, and involve an immediate reaction to a salient situation. Appetitive conditioning procedures also have been used to dissociate similar functions of the CeN and B L A (Hatfield et al., 1996; Parkinson et al., 2000). Interestingly, these nuclei have been implicated in the modulation of mesoaccumbens D A transmission in the context of the same appetitive conditioning paradigms (Robledo et al., 1996; Everitt et a l , 1999, 2000). This raises the possibility that successful behavioral response strategies may involve different mechanisms of mesoaccumbens D A modulation by the CeN and B L A . Everitt et al. (1999; 2000) suggest that theories of amygdala function should account for different roles of amygdala nuclei in controlling adaptive behavioral 26 responses. They also propose that interactions between the CeN and mesoaccumbens D A system in the V T A are necessary for reflexive, Pavlovian conditioned responses, such as autoshaping and freezing behavior. More flexible responses, demonstrated with conditioned reinforcement paradigms, are reliant on interactions between the B L A and NAc. The present data show that stimulation of the CeN does not have an effect on D A efflux in the NAc, but its activation may cause generalised short-lasting changes in mesoaccumbens D A tone. In contrast, stimulation of the B L A induces a long-lasting increase in D A efflux in the NAc. Additional data from our laboratory suggest that the B L A can potentiate its own inputs to the NAc by directly inducing a long-lasting increases in N A c D A efflux (Floresco et al., 2001a). 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Neuroscience 14:427-453. 36 Figure Captions. Figure 1. Different effects of high frequency stimulation of the B L A and CeN on D A efflux in the NAc. A. D A efflux in the NAc measured by microdialysis expressed as percent change from baseline. High frequency stimulation of the B L A (20 Hz, 10 sec; SI) induced a long-lasting significant increase in DA efflux in the NAc (n=7). Two hours later, a second train of stimulation (20 Hz, 10 sec; S2) replicated this effect. Asterisks denote significant differences from baseline (white diamond) at the level of p<0.05. Data points represent a mean level of D A obtained over a 10-min sampling period (see methods for details). Error bars represent S E M (for both A and B). B. Changes in N A c D A efflux expressed as a percent change from baseline measured by microdialysis. High frequency stimulation of the CeN at four different current intensities (S7=300 p. A , 52=450 uA, 53=600 uA, 54=800 uA) failed to induce significant changes in D A efflux (n=8). See Fig. 4 for a summary of histology. Figure 2. Reverse dialysis of iGluR antagonists into the NAc blocks the increase in N A c D A efflux observed following B L A stimulation. A. Stimulation of the B L A (20 Hz, 10 sec, SI) induces a significant increase in D A efflux in the NAc. Two hours later, reverse dialysis of the N M D A receptor antagonist A P - V (100 uM) blocks the expected increase in D A efflux following stimulation of the B L A (n=7, 20 Hz, 10 sec, S2). B. Reverse dialysis of the AMPA/kainate receptor antagonist D N Q X (100 uM) blocks the expected increase in D A following stimulation of the B L A (n=8, 20 Hz, 10 sec, S2) that occurred before drug perfusion began (SI). Both A and B: Asterisks denotes a significant difference from baseline (white diamonds) at the level of p<0.05, error bars represent 37 S E M . The horizontal bars represent the time period during which A P - V (A) and D N Q X (B) were administered into the N A c by reverse dialysis. Figure 3. Effects of intra-VTA Lido infusion on NAc D A efflux evoked by stimulation of the B L A . A. Stimulation of the B L A (20 Hz, 10 sec; 57) induces a significant increase in D A efflux (20 ± 5%) in the NAc of the "Stim" group (white diamonds, n=9) but not in the "Ctrl" (black squares, n=7) or "StimCtrl" (black triangles, n=6) groups. Histological analysis revealed that the electrodes of the Stim group were only located in the caudal B L A (see results section). Eighty minutes later, Lido was infused into the V T A of all groups, resulting 10 min later in a significant 30 ± 7% decrease in basal D A levels in all groups. Immediately following collection of these data, the Stim and StimCtrl groups were stimulated a second time (20 Hz, 10 sec; S2, large data points). Following stimulation, D A levels in the NAc of the StimCtrl group fell to a level not significantly different from the Ctrl group's (53 ± 4% below baseline). D A levels in both of these groups remained significantly below baseline levels for an additional 10 minutes before returning to baseline. In dramatic contrast to the Ctrl and StimCtrl groups, N A c D A efflux in rats of the Stim group increased significantly following the second stimulation. D A levels of the rats in the Stim group were significantly higher than those of the Ctrl and StimCtrl groups for 10 min following the second stimulation (these data are summarized in part B of this figure). Error bars represent SEM. Asterisks denote a significant increase in D A levels above baseline (Stim group, dark circle; Ctrl and StimCtrl groups, white square and white triangle, respectively; p<0.05). Crosses (+) indicate a significant decrease in D A levels from baseline (p<0.05). Number signs (#) 38 indicate a significant difference between groups exist (p<0.05). B. Summary of D A levels for the Stim (black bars), Ctrl (white bars), and StimCtrl (hatched bars) groups following the first stimulation (SI), intra-VTA Lido infusion (VTA-Lido), and the first sample taken after the second stimulation (S2). See caption A for details. Asterisks denote a significant difference between groups (p<0.05). Figure 4. Histology. A. Representative placements of electrodes aimed at the B L A that were (left side) and were not (right side) effective in inducing an increase in D A efflux in all experiments. For clarity not all placements are shown. On the left side, a total of 12 and 13 electrodes were centered in the area of the B L A shown by the plotted placements at -3.3 mm and -3.6 mm, respectively. Black circles correspond to placement of the electrode tip in both A and B. B. Placements of electrodes aimed at the CeN. C. Representative placements of microdialysis probes aimed at the NAc for all experiments in this paper. Black bars are scaled to 2 mm in length. As such, they accurately represent the area of the brain from which each probe sampled. D. Placements of the micro-infusion needles used in the VTA-Lido experiment. A l l figures: Numbers correspond to mm from bregma. Coronal brain sections were adapted from Paxinos and Watson (1997). f Fig. 1 39 A BLA Stimulation B Time (X10 min) C e N Stimulation X 40 -I 3 ifc 30 -LU < 20 -a c 10 -0) c o CO Ch -10 --20 --S1 i S2 S3 S4 ~i i i i i i i i i i i—i—i—i—i—i—i—i—i—i—i—r 3 5 7 9 11 13 15 17 19 21 23 Time (X10 min) —i—i—i—i 25 27 40 A 100 jl/M AP-V -20 "I i i i i i i i i i i i i i — i — i — i — i — i — i — i — i — i — i — i 1 3 5 7 9 11 13 15 17 19 21 23 25 Time (X10 min) B 100/JM DNQX -20 " i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i 1 3 5 7 9 11 13 15 17 19 21 23 25 Time (X10 min) 41 A Intra-VTA Lido Infusion S1 VTA Lido S2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Time (X 10 min) SI VTA Lido S 2 2.80 mm 42 +2.20 mm +1.70 mm +1.20 mm •1.80 mm -2.12 mm •2.30 mm •2.56 mm 4.52 mm 

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