Behavioral/Systems/Cognitive

Aversive Stimuli Differentially Modulate Real-TimeDopamine Transmission Dynamics within the NucleusAccumbens Core and Shell

Aneesha Badrinarayan,1 Seth A. Wescott,1 Caitlin M. Vander Weele,2 Benjamin T. Saunders,2 Brenann E. Couturier,2

Stephen Maren,1,2 and Brandon J. Aragona1,2

1Neuroscience Graduate Program and 2Department of Psychology, University of Michigan, Ann Arbor, Michigan 48109-1043

Although fear directs adaptive behavioral responses, how aversive cues recruit motivational neural circuitry is poorly understood.Specifically, while it is known that dopamine (DA) transmission within the nucleus accumbens (NAc) is imperative for mediatingappetitive motivated behaviors, its role in aversive behavior is controversial. It has been proposed that divergent phasic DA transmissionfollowing aversive events may correspond to segregated mesolimbic dopamine pathways; however, this prediction has never been tested.Here, we used fast-scan cyclic voltammetry to examine real-time DA transmission within NAc core and shell projection systems inresponse to a fear-evoking cue. In male Sprague Dawley rats, we first demonstrate that a fear cue results in decreased DA transmissionwithin the NAc core, but increased transmission within the NAc shell. We examined whether these changes in DA transmission could beattributed to modulation of phasic transmission evoked by cue presentation. We found that cue presentation decreased the probability ofphasic DA release in the core, while the same cue enhanced the amplitude of release events in the NAc shell. We further characterized therelationship between freezing and both changes in DA as well as local pH. Although we found that both analytes were significantlycorrelated with freezing in the NAc across the session, changes in DA were not strictly associated with freezing while basic pH shifts in thecore more consistently followed behavioral expression. Together, these results provide the first real-time neurochemical evidence thataversive cues differentially modulate distinct DA projection systems.

IntroductionFear-evoking cues in the environment powerfully shape our be-havior. While there is an extensive literature surrounding howfearful cues engage the amygdala and related circuitry to generatereactive defensive behaviors (for review, see (LeDoux, 2000; Papeand Pare, 2010; Maren, 2011), there has been less investigationregarding how fearful cues impact motivational neural circuitry.This is notable because fear can be highly motivating. For exam-ple, it is adaptive for fearful stimuli to alter motivated behavior orfoster active avoidance responding (Kelley et al., 2005). It is wellknown that appetitive goal-directed behavior is mediated by pha-sic dopamine (DA) transmission within mesolimbic systems(Mirenowicz and Schultz, 1996; Schultz, 2007; Wanat et al.,2009), and mesolimbic DA is also critical for processing aversiveinformation (Pezze and Feldon, 2004; Faure et al., 2008; Fadok etal., 2009, 2010; Darvas et al., 2011; Valenti et al., 2011; Zweifel et

al., 2011). For example, DA transmission within the NAc is crit-ical for the modulation of conditioned fear, demonstrated byaversive latent inhibition(Young et al., 1993; Gray et al., 1997).However, the specific involvement of phasic DA transmission inaversive motivation remains a matter of considerable debate.

While early studies suggested that aversive cues strictly de-crease DA neuron firing (Mirenowicz and Schultz, 1996; Unglesset al., 2004), more recent studies suggest that anatomically heter-ogeneous subpopulations of DA neurons respond with both in-creases and decreases in firing to aversive stimuli (Brischoux etal., 2009; Matsumoto and Hikosaka, 2009; Bromberg-Martin etal., 2010a,b). These subpopulations contribute to neurochemi-cally and functionally distinct projection systems that are difficultto study electrophysiologically (Bannon and Roth, 1983; Deutchand Roth, 1990; Di Chiara and Bassareo, 2007; Ikemoto, 2007;Belin and Everitt, 2008; Liu et al., 2008; Aragona et al., 2009).Here, we use fast-scan cyclic voltammetry (FSCV) to measuresubsecond fluctuations in DA concentration within distinct me-solimbic projection systems (Ikemoto, 2007; Lammel et al.,2008), terminating in either the nucleus accumbens (NAc) coreor shell while freely behaving rats were presented with a fear-evoking cue. Although FSCV has been used previously to exploreregional differences in DA transmission in response to both cuedand unconditioned appetitive stimuli (Aragona et al., 2009;Brown et al., 2011), studies that have used FSCV to explore aver-sive stimulus processing have focused on primary aversive stimuli(Roitman et al., 2008; Budygin et al., 2012). However, cues and

Received July 24, 2012; revised Sept. 6, 2012; accepted Sept. 8, 2012.Author contributions: A.B., S.M., and B.J.A. designed research; A.B., S.A.W., C.M.V.W., B.T.S., and B.E.C. per-

formed research; A.B. and C.M.V.W. analyzed data; A.B. and B.J.A. wrote the paper.This work was supported by National Institutes of Health (R01MH065961, S.M.), National Science Foundation

(0953106, B.J.A.), and a Rackham Regents Fellowship (A.B.). We wish to thank Drs. Joshua Berke, Richard Keithley,Jeremy Day, Caitlin Orsini, and Vedran Lovic as well as Kirsten Porter-Stransky, Katherine Prater, Brandy Radem-acher, and Ramzy Khabbaz for technical assistance and comments during the assembly of this manuscript.

Correspondence should be addressed to Dr. Brandon Aragona, Department of Psychology, University of Michi-gan, 530 Church Street, Ann Arbor, MI 48109-1043. E-mail: aragona@umich.edu.

DOI:10.1523/JNEUROSCI.3557-12.2012Copyright © 2012 the authors 0270-6474/12/3215779-12$15.00/0

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the unconditioned stimuli they predict drive differential re-sponses in DA neurons (Matsumoto and Hikosaka, 2009). Thus,DA transmission dynamics within motivational circuits underly-ing learned aversive cues must be elucidated.

Here, we provide the first real-time electrochemical data dem-onstrating that a fear cue causes regionally specific changes inboth phasic DA transmission and local pH levels. Presentation ofthe aversive cue decreases DA transmission and causes a basic pHshift within the NAc core but increases DA transmission withoutaltering pH levels within the NAc shell. Together, these findingssignificantly extend our understanding of how aversive cues re-cruit motivational circuits, speaking to a long-standing contro-versy regarding how aversive stimuli alter DA transmission.

Materials and MethodsAnimals. Twenty-two male Sprague Dawley rats weighing 280 –320 gwere used as subjects (Core Conditioned: n � 5; Shell Conditioned: n �6; Core Control: n � 6; Shell Control: n � 5). Rats were individuallyhoused with food and water available ad libitum, and were maintained ona 14 –10 h light/dark cycle. All experiments were conducted during theanimals’ dark cycle. All procedures were performed in accordance withguidelines approved by the University of Michigan University Commit-tee on Use and Care of Animals.

Surgery. Before behavioral manipulations, rats received FSCV surgery(Aragona et al., 2008, Day et al., 2010; Wheeler et al., 2011; McCutcheonet al., 2012). Rats were anesthetized with ketamine (90 mg/kg, i.m.) andxylazine (10 mg/kg, i.m.). A guide cannula (Bioanalytical Systems) wasplaced over the NAc core (anteroposterior (A/P) � 1.7, mediolateral(M/L) �1.3; relative to bregma) or NAc shell (A/P � 1.7, M/L �0.8;relative to bregma) and an Ag/AgCl reference electrode was implanted incontralateral cortex (A/P �0.8, M/L �4.0; relative to bregma). A bipolarstimulating electrode (Plastics One) was positioned over the ventral teg-mental area (VTA; A/P � 3.8, M/L �0.8; relative to the intra-aural line)and secured in a dorsoventral (D/V) position that yielded maximumelectrically stimulated (60 pulses, 60 Hz, 120 �A) DA release within theventral striatum (typically �8.6 mm D/V relative to brain surface). Thestimulating electrode was used at the end of behavioral testing (see be-low) to evoke a range of electrically stimulated DA release magnitudesneeded for statistical analysis; i.e., to construct “training sets” needed toperform principal component analysis (Heien et al., 2005; Keithley et al.,2011).

Behavioral testing. Following surgery, rats were allowed 5–7 recoverydays before receiving pavlovian fear conditioning. The conditioning andextinction phases of the behavioral testing were conducted in distinctobservation chambers (Med Associates) that differed in visual and tactilecues. Consistent with previous studies (Orsini et al., 2011), Pavlovian fearconditioning consisted of five pairings of an auditory stimulus (condi-tioned stimulus [CS] 10 s, 2 kHz, 80 dB) coterminating with an aversivefootshock (unconditioned stimulus [US]; 2 s, 1 mA) with a 60 s intertrialinterval. Control rats received either tone- or footshock-alone training(Arcediano et al., 2005) to control for nonassociative factors related toconditioning. Freezing served as the behavioral index of conditional fearduring both the conditioning and expression sessions. During condi-tioning, behavioral chambers rested on load-cell platforms that re-corded chamber displacement in response to animals’ locomotoractivity. One minute after the final shock presentation, animals weretransported back to their home cages and �24 h later, rats weretransported to a novel environment for voltammetric measures dur-ing a tone-alone test session.

The tone-alone test session, or tone-test, was conducted in a modifiedMed Associates chamber that allowed for voltammetry recordings (Ara-gona et al., 2009). Before behavioral testing, an appropriately sensitiveelectrode was selected (see below). Once an electrode that detected nat-urally occurring phasic DA release events (i.e., transients) was identified(see below), we performed a “context switch,” which entailed turningwhite room lights off and red room lights on, and briefly moving the ratfrom the chamber to remove a padded floor to reveal a grid floor. This

context switch was necessary to reestablish robust locomotor behaviorthat ensured the ability to detect cue-evoked freezing. Immediately fol-lowing the context change voltammetry recordings commenced. After a3 min baseline period, rats were given 15 tone-alone presentations (10 s,2 kHz, 80 dB) with a 1 min intertrial interval. Behavior was recorded andhand scored off-line by experimenters blind to condition (core or shelland control or conditioned). At least two experimenters provided behav-ioral scoring and achieved an inter-rater reliability of 95%.

FSCV. During behavioral conditions described above, FSCV was usedto measure phasic changes in DA and local pH. For each subject, a freshglass-encased carbon-fiber microelectrode was lowered into the NAccore or shell (Phillips et al., 2003a; Aragona et al., 2008). Electrode con-struction and calibration were conducted in the same manner as de-scribed in our previous studies (Aragona et al., 2009), with the additionaluse of a novel flow cell, which was used for in vitro calibrations (Sinkala etal., 2012). Epoxy was used to create a more robust and reliable sealbetween the carbon fiber and glass casing to ensure clean and stablerecordings. Electrodes were positioned within the NAc core or shellwhere naturally occurring DA transients were reliably detected, as re-ported in many previous FSCV studies (this is now a standard operatingprocedure) (Wightman et al., 2007; Aragona et al., 2008; Roitman et al.,2008). Consistent with previous studies, if the electrode did not detect ahigh DA “transient” frequency, it was withdrawn and another inserteduntil DA transients were reliably detected (Aragona et al., 2008). Weselected highly sensitive electrodes across all subjects as previous studieshave shown that this is necessary to detect decreases in transient fre-quency and the associated decrease in time averaged [DA] (Roitman etal., 2008; Aragona et al., 2009). Once stable DA transients were detected,the environmental context was altered by changing the visual and tactileelements of the chamber, as described above, and FSCV recordings be-gan. Following FSCV recordings during the behavioral study, DA releasewas evoked in the freely moving subject by electrical stimulation of theventral tegmental area (Phillips et al., 2003a; Stuber et al., 2005) for use inprinciple component analyses to convert the recorded current into [DA]and pH units (Heien et al., 2004; Keithley et al., 2011).

To verify the identity of the electro-active analytes, current values wereconverted to [DA] and pH units using principal component analysis(Heien et al., 2004; Keithley et al., 2011). Detection and analysis of DAhave been described in detail previously (Heien et al., 2005; Aragona etal., 2008). Consistent with previous FSCV studies (Day et al., 2007; Ara-gona et al., 2009), mean changes in [DA] were calculated relative to a zeropoint determined by the background subtraction applied to the raw data.Background subtraction was performed at the lowest current valuewithin the 10 s preceding cue onset separately for each tone presentation(Roitman et al., 2008; Aragona et al., 2009). Phasic changes in [DA] weredetermined within a 30 s sampling period that was centered around toneduration, with a 10 s baseline, 10 s recording during CS presentation, anda 10 s post-CS period. Transients were identified using MiniAnalysis(Synaptosoft) as previously described (Aragona et al., 2008). A transientwas defined as a fivefold or greater increase in [DA] relative to the rootmean square noise value taken from the same electrode (Heien et al.,2005).

Characterization of DA and pH changes. FSCV allows detection of localchanges in pH that frequently accompany DA transmission (Venton etal., 2003; Heien et al., 2005; Roitman et al., 2008) and these two analytesare easily distinguished by cyclic voltammogram (CV) analysis, in whichbackground subtracted changes in current detected at the carbon-fiberelectrode are plotted against the applied potential (i.e., the voltage rampfrom �0.4 V to 1.3V and back; described in detail elsewhere; Heien et al.,2005) and time. For each time point, the change in current (resultingfrom the oxidation and reduction of the neurochemical species) as afunction of the applied potential may be determined. This represents theCV for a given neurochemical species and the shape of the CV reveals itsidentity (Fig. 1a, i–iii, insets) (Phillips et al., 2003a; Heien et al., 2005).This is conveniently visualized in color plots, wherein changes in currentare plotted in false color against both time and applied voltage.

Electrical stimulation of DA afferents reliably alters both DA releaseand local changes in pH (Phillips et al., 2003a; Heien and Wightman,2006). In this example (Fig. 1a), two naturally occurring DA transients

15780 • J. Neurosci., November 7, 2012 • 32(45):15779 –15790 Badrinarayan et al. • Dopamine Transmission during Fear Expression

(at random, unpredictable times) were detected (emphasized by invertedwhite triangles) before electrical stimulation. The CV corresponding tothe second transient is shown above the color plot (Fig. 1a, i) and its peakmagnitude is shown in the [DA] trace (Fig. 1b). Upon electrical stimula-tion of dopaminergic afferents, a near instantaneous increase in currentis detected at the peak oxidational potential for DA (�0.65 V of thevoltage ramp; Fig. 1a, ii to inspect the CV of electrically evoked DArelease). Current at this point in the voltage ramp (Fig. 1a, black line) isconverted to [DA] [Figure 1b; described in detail elsewhere (Heien et al.,2005; Keithley et al., 2011)]. In contrast to electrically evoked changes inDA transmission, local changes in pH follow electrical stimulation by adelay of several seconds (Fig. 1a,b) and its peak change in current isprimarily evident at �0.4 V in the applied waveform (Fig. 1a, iii). Con-version from current to pH units (Fig. 1b, red line) has been described indetail elsewhere (Heien et al., 2005; Roitman et al., 2008).

Current changes that are identified as DA or pH, based on statisticallysignificant concordance between the CVs of naturally occurring signalsand CVs of electrically evoked signals, were converted into concentrationmeasures using calibration factors obtained from in vitro electrode char-acterization (Fig. 1c–f ). Numerous electrodes were tested against a seriesof known DA (Fig. 1c,d) and pH values (Fig. 1e,f ) to determine theaverage change in current following administration of known values of[DA] or pH units. The slopes of these resulting curves were subsequentlyused as the calibration factor for converting analyte-evoked current into[DA] or pH units. Consistent with previous reports (Roitman et al., 2008;Aragona et al., 2009), there were no differences in stimulated release ofDA or pH between the NAc core and shell, as seen in representative tracesfrom each region (Fig. 1g–j). It should be noted that, as with all FSCVstudies, these DA and pH values are �[DA] and pH over the lowestcurrent point in each trace. Thus, they do not represent absolute levels ofDA or pH, but rather changes over an arbitrary zero point.

Percentage change in [DA] was calculated for each tone presentationover a 30 s sampling window (10 s pretone period, 10 s tone presentation,and 10 s post-tone period) as follows:

% Change �

Average ��DA� following tone presentation� Average ��DA� preceding tone presentation

Average ��DA� during pre tone period* 100.

Similarly, fold-change in pH units was calculated for each tone pre-sentation as follows:

Fold Change in pH �Average �delta pH units following tone presentation

Average �delta pH units during pre tone period.

Here, “following tone presentation” indicates the time period includ-ing the tone presentation and the post-tone period, or the 20 s followingtone onset.

Histology. Following behavioral and neurochemical testing, animalswere euthanized with an overdose of ketamine. To allow for histologicalverification of electrode placements, either electrolytic lesions were madeat the same microdrive setting used for the experiment (Fig. 1) or anotherestablished method was used wherein the A/P and M/L coordinates arevery clearly identified and the D/V coordinate was “reconstructed” basedon the known relationship between the settings of the microdrive andhow far this lowers the electrode (Robinson et al., 2002).

Statistics. Analysis of freezing, both between groups and across thesession was performed using two-way repeated-measures ANOVA withBonferroni corrected post hoc tests. Changes in [DA] as well as pH unitswere extracted using principal components regression as previously de-scribed (Heien et al., 2004; Aragona et al., 2008; Keithley et al., 2011).CS-evoked changes in [DA] calculated as percentage change in [DA]following tone onset and transient amplitude were assessed using one-way repeated-measures ANOVA and Bonferroni corrected post hoc com-parisons. Binned [DA], pH, and transient probability data were analyzedusing a linear-mixed model regression (Aragona et al., 2008) due to itssuperior ability to handle repeated-measures data (Verbeke, 2009). Forthese analyses, post-tone bins were compared with a pretone baselinevalue with time as a repeated fixed factor and region as a fixed factor.

Changes in [DA], pH, and transient frequency and amplitude were cal-culated in five-tone blocks, and statistical analyses were performed on theresulting data. All data are presented as mean SEM unless otherwisenoted. A p value � 0.05 was considered significant. Linear mixed-modelregressions were performed in SPSS version 19 for Windows; all otherstatistics were performed in Prism version 5.0 for Mac OSX.

ResultsHistology and behaviorOne day following fear conditioning, rats were moved to a novelenvironment and FSCV was used to measure real-time DA trans-mission (Fig. 1) within the NAc core (Fig. 2a,c) or shell (Fig. 2b,c)(Rebec et al., 1997; Robinson and Wightman, 2004; Aragona et al.,2009). Three minutes after the context shift, rats were presented with15 CS-alone presentations (at 1 min intervals) while real-time neuro-chemical measurements were continued. Freezing behavior did not dif-fer between conditioned groups (F values � 1.0, Fig. 2d) but wassignificantly higher than controls (main effect of group, F(1,15) � 332.6,p� 0.001) until the final block of tones during which there was no dif-ferenceinbehaviorbetweenconditionedandcontrolanimals(p�0.05;Fig. 2d). Rats that had previously received paired CS–US presentationsshowed nearly 100% freezing behavior during block 1 (first five tonepresentations)(Fig.2d).Duringthenextblockoffivetonepresentations(block 2), freezing behavior began to decline and returned to baselinelevels by the third block of tone presentations (Fig. 2d). Control ratsthat received only tone (CS-alone) or shock (US-alone) presentationtraining did not show freezing behavior during the tone test (Fig.2d). It should be noted that control animals show a slight upwardtrend in freezing toward the end of the session; however, this was notsignificantly different from freezing levels earlier in the session andwas not likely due to fear-evoked freezing responses but rather gen-eralized decreases in locomotor activity in unconditioned animalsfollowing a prolonged period of time sitting within the chamber.Histological verification of electrode placements was performed atthe end of the experiment (see Materials and Methods; Fig. 2a–c).

Fear-evoking cues diminished DA release within the NAc corebut enhanced DA release within the NAc shellAfter appropriate electrodes were selected, real-time [DA] and localpH changes were measured across the context switch and through-out the behavioral testing session. Although previous FSCV studieshave demonstrated that presentation of novel stimuli (Robinson andWightman, 2004) and entry into novel environments can increaseDA transmission (Rebec et al., 1997), this context change did notsignificantly alter phasic DA transmission (transient frequency dur-ing final 3 min before context switch � 10.68 0.60 and during theinitial 3 min following the context switch � 10.81 0.65; t(10) ��196, p � 0.85). This baseline measure of transient frequency isconsistent with previous FSCV studies (Cheer et al., 2004; Aragona etal., 2008; Sombers et al., 2009). Indeed, the detection of basaltransients before experimental manipulation (Wightman etal., 2007) is essential to assure electrode fidelity and increasesconsistency across experimenters. Moreover, it is essential toallow decreases in DA transmission to be detected using FSCV(see below). These, among other critical features of basal tran-sient detection, have contributed to it becoming a standard op-erating procedure in FSCV data acquisition when acute electrodeimplantation is employed on the day of the experiment.

Although rats in both the core-conditioned and shell-conditioned groups showed similar behavioral patterns duringthe tone test (Fig. 2d), presentation of the CS caused dramaticallydifferent neurochemical changes within these NAc subregions(Figs. 3, 6). For analysis of fear-evoked changes in [DA], we con-

Badrinarayan et al. • Dopamine Transmission during Fear Expression J. Neurosci., November 7, 2012 • 32(45):15779 –15790 • 15781

structed heatmaps to examine how aver-age [DA] changed with CS presentationacross the session in both the core (Fig. 3a;n � 5) and the shell (Fig. 3b; n � 6).Within the NAc core, cue presentationcaused a rapid and prolonged decrease in[DA] (Fig. 3a). Quantification of the per-centage change in CS-evoked �[DA]within the NAc core showed that CS pre-sentation dramatically decreased [DA]within conditioned, but not control, sub-jects only within the first block of trials(Fig. 3c). Two-way ANOVA analysis ofcore-conditioned versus core controlgroups revealed a significant interaction be-tween time and group (F(2,16) � 17.3, p �0.01). Post hoc tests revealed a significant dif-ference between core-conditioned animalsand core-control animals only during block1 (i.e., the first five trials, p � 0.01). We nextanalyzed the resulting concentration data in2 s bins for each block of trials, collapsedacross five tones in each of our behaviorallydesignated blocks for both the NAc core andshell. Within the NAc core, CS-presentationcaused a significant decrease in [DA] rela-tive to the pre-CS period that was rapid andlong lasting selectively during block 1 (Fig.3d; F(10, 23.7)�10.32, p � 0.01 for block 1).No significant changes in [DA] were ob-served during block 2 (Fig. 3e; F(10, 31.4) �1.0, p � 0.71) or block 3 (Fig. 3f; F(10, 32.5) �1.0, p � 0.73).

In stark contrast to the NAc core, CSpresentation increased [DA] within theNAc shell, although still selectively withinthe first block of trials (Fig. 3b,g); note therobust average increase during block 1 de-picted in the heat map. Quantification ofpercentage changes shows a main effect ofgroup (F(2,32) � 10.6, p � 0.01) and posthoc tests confirm that [DA] was signifi-cantly increased within the NAc shell inconditioned, but not control, animalsonly within the first block of trials (Fig. 3g;p � 0.05). Heatmaps illustrate the “dif-fuse” (i.e., poorly time locked to the CS)increase in the NAc shell following CSpresentation early during fear expres-sion (Fig. 3b). However, the increase wasstrongest at cue offset. Specifically, analy-sis of binned concentration data revealedan increase in [DA] following CS presen-tation during block 1 that was only signif-

Figure 1. Changes in [DA] and pH values are characterized based on stimulation both in vivo and in vitro. a, Representative colorplot showing current changes observed at the recording electrode plotted against the applied voltage and time. Analytes (DA andpH) are identified based on specific oxidation-reduction profiles. The holding potential applied to the carbon-fiber electrode (�0.4V) was ramped to �1.3 V and back to 0.4 V at a rate of 10 Hz. This example shows two spontaneous DA transients, indicated bywhite inverted triangles. Electrical stimulation (24 pulses, 60 Hz) of the VTA results in a near instantaneous surge in [DA] and a moredelayed and long-lasting basic shift in pH. Vertical dashed lines on the color plot indicate the time point of representative cyclicvoltammograms, shown above. Horizontal black (DA) and red (pH) lines indicate the applied voltage at the point of maximumoxidation, used to identify neurochemical species. b, Traces for �[DA] and pH units were obtained by using principal componentsanalysis. �[DA] for the first transient observed in a and the stimulation is shown, as well as peak change in pH units. c, Calibrationcurve for DA. Known concentrations of DA were used to determine the average current evoked in a standard set of electrodes. Theslope of the resulting trend line was then used as the calibration factor for conversion of current to DA concentration. d, Represen-tative color plots and current traces recorded in vitro during the application of specific DA concentrations. Blue bars indicate infusionof DA. e, Calibration curve for pH. Current changes were again recorded for known basic shifts in pH. The resulting slope was again

4

used as the calibration factor. f, Representative color plots andcurrent traces recorded in vitro during the application of basicpH shifts. Blue bars indicate the duration of the infusion. g–j,Representative color plots and traces of electrical stimulation(24 pulses, 60 Hz) of DA afferents to the NAc core (g, i, j) andshell (h–j). These plots indicate similar DA and pH responseswithin the core and shell.

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icant during the final five bins (i.e., following tone offset, p �0.05, Fig. 3h). However, we still refer to this as a diffuse increasebecause it was not nearly as robustly or reliably associated withany temporal attributes of cue presentation, compared with thatseen within the core. However, it should be noted that the first binshowing a significant increase in [DA] was the bin immediatelyfollowing cue offset. This effect, however, is an average effect andwas not consistently observed across subjects, and animals showedno difference in behavior at cue offset (i.e., they still showed robustfreezing during early CS presentations). Taken together, this sug-gests that the increase is unlikely to serve as an emotional “relief”signal at the omission of an expected aversive footshock (Ikemotoand Panksepp, 1999).

No significant changes in [DA] were observed during block 2or block 3 (Fig. 3i,j; F � 1.0, p � 0.05). Importantly, there wasalso a lack of a CS-evoked response in control rats (Fig. 3c,g). Thissuggests that the changes in [DA] in conditioned animals weredue to the CS–US association and not to sensitization of the DAsystem due to prior experience with the tone or shock. This isconsistent with previous reports from other groups that uncon-ditioned tone presentations do not evoke changes in dopaminerelease (Phillips et al., 2003b; Robinson and Wightman, 2004).

Cue-evoked changes in DA release within both the NAc coreand shell can be attributed to altered phasic transmissiondynamicsTo determine whether aversive cue-evoked changes are mediatedby specific changes in phasic DA transmission dynamics, we as-sessed how the aversive CS altered the probability and magnitudeof DA transients. In FSCV measures, average [DA] is comprisedof time-averaged DA transients (Roitman et al., 2008) superim-posed on basal extrasynaptic [DA] levels that are not detected bythis technique (Venton et al., 2003; Arbuthnott and Wickens,2007). As described previously (Aragona et al., 2008, 2009) weanalyzed the probability of the occurrence of transients to ap-

proximate changes in the number of pha-sic release events and transient amplitudeto estimate changes in the magnitude ofDA released.

A representative color plot and DAconcentration trace within the NAc core(Fig. 4a) and shell (Fig. 4d) following thepresentation of an aversive CS shows thatbefore CS presentation, transients wereobserved consistently during the baselineperiod in both the NAc core and shell(baseline probability: core � 0.55 0.07and shell � 0.63 0.04, t(9)�0.02,p�0.9). Within the NAc core, CS presen-tation caused a significant decrease intransient probability (F(10, 25.8) � 6.51,p � 0.001; Fig. 4a,b), suggesting that theCS-evoked decrease in [DA] within theNAc core is due to a decrease in the occur-rence of phasic release events. In contrast,presentation of the same CS did not altertransient probability within the NAc shell(Fig. 4d,e) (F(10, 30.3) � 1.67, p � 0.14).Analysis of transient amplitude, on theother hand, showed that while CS presen-tation did not alter release magnitudewithin NAc core (t(5) � 0.78, p � 0.48; Fig.4c), there was a significant increase in

transient amplitude (t(5) � 9.01, p � 0.01) following CS onsetwithin the NAc shell (Fig. 4f). These data suggest the CS-evokedincrease in [DA] described above within the NAc shell is due to acue-evoked increase in the magnitude of phasic DA releaseevents. Furthermore, it is important to note that, with FSCV, onlyacute, background subtracted effects are seen. Thus, it is possiblethat the phasic increase in the shell we report here may be anunderestimation of the overall increase in [DA] occurring withinthe NAc shell, due to the differential nature of the technique.

Fear-evoked changes in DA transmission in the NAc core butnot shell are accompanied by basic shifts in pHAs CS-related changes in DA transmission in the both the NAccore and shell were selective to the first five-tone block, furtheranalysis focused on this block of CS presentations. Subregiondifferences in the neurochemical responses to aversive cues areobvious upon inspection of average color plots for the NAc core(Fig. 5a) and shell (Fig. 5b) for the first block of CS presentations.Before CS onset, [DA] and pH levels were equivalent within theNAc core and shell (DA: t(51) � 0.16, p � 0.87; pH: t(50) � 0.51,p � 0.63) (Fig. 5c,d). However, at CS onset, [DA] rapidly de-creased within the NAc core (Fig. 5a,b; statistically described else-where) and this decrease was followed by a long-lasting basic pHshift (Fig. 5a,d; t(23) � 2.62, p � 0.05). Within the NAc shell, it isevident that the increase in [DA] by the fear CS (Fig. 3) is quitediffuse and robustly not time locked to tone presentation (Fig.5b,c). Moreover, in contrast to the NAc core, there was no changein local pH within the NAc shell following presentation of theaversive cue (t(27) � 0.73, p � 0.47)(Fig. 5b,d). These differencesin core-shell pH shifts were not attributable to differences inoverall pH responsiveness between these regions (Fig. 1g–j; Roit-man et al., 2008). Thus, presentation of the fear CS alters local pHselectively within the NAc core, which is the region in whichcue-evoked decreases in DA transmission are strongly timelocked to stimulus presentation. A similar relationship between a

Figure 2. Histology and fear expression during recording sessions. a, Example of histological verification of recording site in theNAc core (b) and within the NAc shell. c, Diagrammatic representation of electrode placements for all animals within the NAc.Core-conditioned placements are indicated in orange (n � 5), Shell-Conditioned placements are indicated in cyan (n � 6), andplacements for control animals are shown in black (n � 11). Although the placements are distributed throughout both cerebralhemispheres here for clarity, recordings from all animals were taken from the same hemisphere. Red indicates the examples shownin a and b. d, Percentage freezing during fear expression for core-conditioned, shell-conditioned, and control animals. There wasno difference between conditioned groups in fear expression. Unconditioned rats did not freeze during CS presentation. Allconditioned rats showed robust freezing that attenuated as the session progressed. By the end of the session, conditioned ratsshowed no more freezing than control rats.

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basic pH shift and a time-locked decrease in [DA] has also beenshown after presentations of an unconditioned aversive tastestimulus (intra-oral infusion of quinine) (Roitman et al., 2008).However, while the quinine-evoked decrease in [DA] was simi-larly associated with a basic pH shift, this neurochemical re-sponse occurred within the NAc shell whereas the aversive CS(present study) resulted in this [DA]/basic pH alteration withinthe NAc core.

To more thoroughly explore how DA and pH changes relatedto fear expression, we analyzed within-trial trajectories of freez-ing for block 1 (Fig. 5e). For each trial, we assessed freezing acrossthe 30 s sampling window used for neurochemical analyses. Thisshowed that freezing has a rapid onset following cue presentationand slow attenuation following cue offset, demonstrating that theneurochemical dynamics that best mirror the behavior occurwithin the NAc core and not the NAc shell.

Cue-evoked basic pH shifts within the NAc core but not shelltrack freezing behaviorGiven that fear cues caused a strictly region-specific shift in localpH, we analyzed this in more detail. Specifically, we (in the same

manner that we used for the [DA] data) compared alterations incue-evoked changes in pH units across blocks of fear expression.

We constructed heatmaps to study how cue-evoked pHchanges within the NAc core and shell evolve across the 15 tonesession (Fig. 6a,b). Within the NAc core (Fig. 6a), cue presenta-tion was followed by a basic shift in pH that lasted through blocks1 and 2 but diminished through block 3. Quantification of thefold-change in pH from the pretone period to the post-tone pe-riod in conditioned animals showed that cue presentation re-sulted in a significant increase in pH (i.e., a basic shift) within theNAc core during both the first and second blocks in conditioned,but not control, subjects (Fig. 6c). Two-way ANOVA analysis ofcore-conditioned versus control groups showed significant maineffects of group (F(1,18) � 53.96, p � 0.001)and time (F(2, 18) �20.10, p � 0.001) and revealed an interaction between group andtime (F(2, 16) � 5.59, p � 0.013). Post hoc tests showed a significantdifference between core-conditioned animals and control ani-mals during both block1 (p � 0.001) and block 2 (p � 0.05). Forblock 3, there was no difference between conditioned and controlsubjects (p � 0.05). We then analyzed each five tone block of pHdata in 2 s bins (Fig. 6d–f). Within the NAc core, cue presentation

Figure 3. Fear cues cause decreased DA transmission in the NAc core but enhanced DA transmission in the NAc shell. a, b, Heatmaps depicting trial-by-trial �[DA] across all 15 cue presentationsin the core (n � 5) and shell (n � 6), respectively. Tone presentation was immediately followed by a dramatic decrease in [DA] within the NAc core, but within the shell, [DA] was gradually increasedfollowing cue offset. In both regions, the effect attenuated as the session progressed. c, Percentage change in mean [DA] in the NAc core in conditioned versus unconditioned animals in response toCS presentations. A significant decrease in [DA] was observed in response to CS presentations during the first five tone block. d–f, �[DA] within the NAc core relative to CS presentation across eachblock of trials. [DA] was calculated in 2 s bins. Tone presentation causes an immediate and significant decrease in [DA] during block 1 (d) that attenuated during subsequent blocks (e, f). g,Percentage change in mean [DA] in the NAc shell in conditioned versus unconditioned animals in response to CS presentations. A significant increase in [DA] was observed in response to the CS duringthe first five tone block. h–j, �[DA] within the NAc shell relative to CS presentation across each block of trials. Tone presentation resulted in a gradual increase in [DA] that was significant relativeto pretone [DA] following cue offset during block 1 (h). There was no significant change following tone presentation in block 2 (i) or 3 (j). *p � 0.05.

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caused a significant basic shift in pH units during both blocks 1(Fig. 6d; F(10, 23.7) � 5.94, p � 0.001) and 2 (Fig. 6e; F(10, 32.7) �6.10, p � 0.001). We observed no significant changes in block 3(Fig. 6f; F � 1.0). It should be noted that basic pH shifts withinthe core are significant during blocks 1 and 2, but not 3, whichmirrors freezing behavior in conditioned animals (Fig. 2d).

In contrast to the NAc core, CS presentation did not alter pHwithin the NAc shell (Fig. 6b). Quantification of the fold-changein pH following tone presentation confirms this observationfrom the heatmaps and shows no changes in pH within the NAcshell in conditioned or control animals (Fig. 6g). This was furtherconfirmed by examining the binned pH data for each 5-tone

Figure 4. Changes in [DA] in response to the fear-evoking CS are attributable to changes in the probability and amplitude of DA release events. a, d, Representative transient traces. Whiteinverted triangles indicate transients. The panel underneath each color plot shows the converted [DA] traces. b, e, Transient probability calculated during 2 s bins for block 1 within the NAc core (i.e.averaged over first five tones for n � 5; 25 traces all together) and shell (i.e. averaged over first five tones for n � 6; 30 traces). Tone onset caused a significant decrease in transient probability thatlasted the duration of the tone presentation, but returned to pretone levels following tone offset in the NAc core. Transient probability did not change with tone presentation in the NAc shell. c, f,Average transient amplitude before and following cue onset in the NAc core and shell. Cue onset did not alter transient amplitude in the NAc core, but increased transient amplitude in the NAc shell.*p � 0.05.

Figure 5. Cue onset is accompanied by basic pH shifts within the NAc core. a, b, Average color plots for the first block of CS presentations in the NAc core (n � 5; a) and shell (n � 6; b) weregenerated to illustrate the raw changes in neurochemical activity associated with tone presentation (gray bar). Within the NAc core, cue onset is accompanied by a delayed but lasting basic pH shift.No visible pH shifts were detected in the shell. c, Quantification of changes in [DA] in core and shell following cue onset during block 1. Cue presentation altered both core and shell [DA]. d,Quantification of pH changes during block 1 within the NAc core and shell. Cue presentation results in a significant basic pH shift within the NAc core, but does not cause any change in pH within theNAc shell. e, Within-trial probability of freezing across block 1 for both core-conditioned and shell-conditioned rats. Cue onset causes rapid increases in freezing probability that last throughout thetone presentation and attenuate slowly following tone-offset.

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block (Fig. 6h–j). No significant cue-evoked changes in pH wereobserved in block 1 (Fig. 6h), block 2 (Fig. 6i), or block 3 (Fig. 6j)within the NAc shell.

Changes in DA transmission are related to but not caused bychanges in locomotor activityAs the NAc has been implicated in the generation of generallocomotor activity, we assessed whether changes in [DA] and pHwithin the NAc core and shell were correlated with the behavioraloutput quantified in this study, freezing. Given that CS-evokeddecreases in [DA] within the NAc core occur consistently onlyduring early trials, when fear expression is high, it is tempting toexpect that there may be a strong, and perhaps causal, relation-ship between changes in [DA] and freezing in this region. How-ever, as demonstrated by a representative rat (Fig. 7a,b), whichshowed equivalent freezing behavior on individual trials withinthe first and second block of trials and yet showed a robust de-crease in [DA] in block 1 (trial 2) (Fig. 7a) and no change in [DA]in block 2 (trial 8) (Fig. 7b), this is clearly not the case. Thissuggests that changes in [DA] were not directly causal with re-spect to changes in behavior.

However, quantification of the relationship between DA andfreezing showed that, despite the absence of a strongly causalrelationship between changes in [DA] within the NAc and freez-ing, there was a relationship between behavioral output and DAneurotransmission. Specifically, there was a significant correla-tion between changes in [DA] and freezing across all trials in the

NAc core (r 2 � 0.23, p � 0.01)(Fig. 7c) and shell (r 2 � 0.07, p �0.05) (Fig. 7e). This is consistent with our general observationthat changes in [DA] were greatest at the beginning of the testsession, when freezing was highest, and extinguished as the ses-sion proceeded.

We also observed a modest but significant correlation be-tween changes in basic pH and freezing in the NAc core (r 2 �0.09, p � 0.05; Fig. 7d) but not the NAc shell (r 2 � 0.03, p � 0.27;Fig. 7f). This relationship between freezing and basic pH changeswithin the NAc core may be of interest because it has been sug-gested that changes in pH correspond to increased postsynapticneural activity, with alkaline changes in pH corresponding withincreased metabolic activity within the structure (Roitman et al.,2008; Ariansen et al., 2012). The correlation between pH andlocomotor activity, taken together with the change in DA trans-mission during the initial block of trials, may indicate that theNAc core is involved in organizing behavioral outputs in re-sponse to aversive stimuli. These data show that changes in DAtransmission only occur in response to early trials, when reorga-nization of behavioral strategies in response to learned fearinformation would be expected, while other measures of neu-ral activity (i.e., pH) may track the behavioral output.

DiscussionThe present study demonstrates, for the first time, that fear-evokingstimuli differentially alter phasic DA transmission across NAc subre-gions.Fearcuesdecreased[DA]withintheNAccorebutincreased[DA]

Figure 6. Fear cues cause basic shifts in pH within the NAc core but not within the NAc shell. a, b, Heatmaps depicting trial-by-trial pH shifts across all 15 cue presentations in the core (n � 5) andshell (n � 6), respectively. Tone presentation was followed by a basic pH shift within the NAc core, but not within the shell. The basic shift within the NAc core remained prominent through blocks1 and 2 and attenuated during block 3. c, Fold-change in average pH in the NAc core in conditioned versus unconditioned animals in response to CS presentations. A significant increase in pH wasobserved in response to CS presentations during the first and second five tone blocks. d–f, �pH units within the NAc core relative to CS presentation across each block of trials. pH was calculated in2 s bins. Cue presentation causes a significant increase in pH during block 1 (d) and 2 (e) that attenuated during the final blocks (f). g, Fold-change in mean pH in the NAc shell in conditioned versusunconditioned animals in response to CS presentations. There was no difference in pH changes within any block between conditioned and unconditioned animals. h–j, �pH within the NAc shellrelative to CS presentation across each block of trials. Tone presentation did not significantly alter pH shifts in the NAc shell during blocks 1 (h), 2 (i) or 3 (j). *p � 0.05.

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withintheNAcshell.Theseregionallyspecificchangesareattributabletochanges in phasic DA transmission, as the aversive cue decreasedthe probability of DA transients within the NAc core but in-creased the magnitude of transients within the NAc shell. More-over, changes in [DA] are not strongly causally attributed to thecessation of locomotor activity. Thus, rather than DA coding aunitary motivational signal, multiple mesolimbic DA systemsmay process distinct aspects of motivated behavior, includingstates associated with fear-evoking cues (Ikemoto, 2007; Aragonaet al., 2009; Bromberg-Martin et al., 2010b; Lammel et al., 2011).

Aversive cues decrease DA transmission specifically in theNAc CoreHere, we demonstrate that conditioned aversive cues evoke arapid and prolonged decrease in [DA] in the NAc core that isattributable to decreased DA release probability. This is consis-tent with many previous studies that have shown that acute aver-sive stimuli cause phasic decreases in DA neuron firing(Mirenowicz and Schultz, 1996; Ungless et al., 2004) and onerecent study demonstrated that the duration of DA neuron inhi-bitions is associated with a behavioral measure of conditioned

fear (Mileykovskiy and Morales, 2011).Although it has been suggested that pha-sic decreases in DA transmission maycode negative motivational value evokedby aversive stimuli (Bromberg-Martin etal., 2010b), the present study provides thefirst evidence regarding the forebrain lo-cus for this decrease. Specifically, dimin-ished DA neuron firing, and thesubsequent decrease in [DA], appears tohave functional effects within the NAccore.

While we know relatively little, mech-anistically, about phasic decreases in DAactivity, recent work demonstrating thatVTA-GABA cells are involved in condi-tioned place aversion (Tan et al., 2012)and in disrupting reward-seeking behav-iors (van Zessen et al., 2012) suggests thatthese cells may play an important role inmodulating DA release to aversive cuesand events. Interestingly, there is compel-ling evidence that aversive motivationalsignals are encoded by habenular cells(Matsumoto and Hikosaka, 2009; Hong etal., 2011) and it has been suggested thattheir inhibitory projections to DA neu-rons may be driven by these VTA-GABAergic cells. Future work will beneeded regarding how interactions amongthese neural populations may allow for theDA signals we observe here to arise.

Importantly, decreased [DA] withinthe NAc core does not appear to beuniquely involved in representing aver-sive stimuli. Previous studies have shownthat aversive taste cues cause phasic de-creases in [DA] within the NAc shell(Roitman et al., 2008; Wheeler et al.,2011). Thus, phasic DA transmissionwithin both the NAc core and shell appearcapable of encoding motivational value.

These core-shell differences in aversive cue modulation of DAmay represent differences in how unconditioned versus condi-tioned stimuli are represented in these circuits. Recent workcharacterizing how phasic DA release within the NAc supportsappetitive motivational signals has demonstrated that while un-conditioned rewards elicit strong DA release within the NAc shell(Aragona et al., 2008, 2009), reward predictive cues preferentiallyincrease DA release within the NAc core (Day et al., 2007; Ara-gona et al., 2009; Wanat et al., 2010; Flagel et al., 2011). Theaversive cues previously used to alter DA transmission within theNAc have been tastants that have caused decreased DA transmis-sion in the NAc shell (e.g., Roitman et al., 2008, Wheeler et al.,2011). Taken together with previous appetitive and aversivework, our data suggest that phasic signaling within the NAc coremay represent a common mode of transmission underlying in-centive motivational responses especially for conditioned stimuli(Robinson and Berridge, 2001; Berridge, 2007). The directional-ity of the shift (i.e., increases or decreases) may encode motiva-tional value, or may rather reflect the organization of behavioralstrategies into active (e.g., reward seeking, avoidance) or passive(e.g., freezing) forms, respectively.

Figure 7. Analysis of the relationship between locomotor activity and neurochemical changes. a, b, Representative traces froma single animal that displayed identical freezing behavior on two trials during block 1 (a) and block 2 (b) illustrate that [DA] doesnot strictly follow locomotor activity. c, Quantification of the correlation between percentage change in [DA] and freezing in theNAc core shows a significant correlation between behavioral expression and changes in dopamine. d, Quantification of the corre-lation between pH and freezing revealed a modest but significant relationship in the NAc core. e, f, Conversely, no relationshipswere observed between changes in [DA] (e) or pH (f) and freezing within the NAc shell.

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Theories of striatal activity posit that the striatum is critical forbehavioral selection, and it has been suggested that enhancedphasic DA signals are critical for identifying and reinforcing ac-tions to organize novel and adaptive behavioral strategies (Red-grave et al., 2008). Likewise, phasic decreases in DA release mayallow reorganization of behavioral strategies to support cessationof any activity that might lead to an aversive outcome via theindirect pathway through the basal ganglia (Kelley et al., 2005;Kravitz et al., 2012). The results presented here are consistentwith these ideas, as the decreases observed here are specific toinitial tone presentations, when behavioral strategies are selectedas well as the period when the animals are experiencing the mostheightened aversive motivational states.

Moreover, the specificity of the decrease to the NAc core, aswell as the fact that the decrease was only present during early CSpresentations, likely explains why this effect was missed by manyprevious microdialysis studies (Wilkinson et al., 1998; Levita etal., 2002; Martinez et al., 2008). The subsecond temporal resolu-tion of FSCV allows for phasic neurochemical changes to be de-tected and therefore better reveals the nature of how DAtransmission is altered by environmental cues in real-time.

Cue-evoked increases in DA transmission in the NAc ShellInterestingly, the current study also demonstrates that aversivecues increase DA transmission in a regionally specific manner.Indeed, many studies spanning multiple techniques have recentlysupported this claim (Brischoux et al., 2009; Matsumoto andHikosaka, 2009; Lammel et al., 2011; Zweifel et al., 2011). Con-sistent with several previous microdialysis studies (Kalivas andDuffy, 1995; Ikemoto and Panksepp, 1999; Pezze et al., 2001;Martinez et al., 2008), the present study demonstrates that pav-lovian aversive cues enhance DA release specifically within theNAc shell. However, it is unclear what this increased [DA] in theNAc shell is encoding. It is well established that phasic increasesin [DA] within the NAc shell are associated with primary rewards(Di Chiara and Bassareo, 2007; Roitman et al., 2008). Thus, it ispossible that increased [DA] following a fearful cue reflects a“relief” signal when the expected footshock is absent (Ikemotoand Panksepp, 1999). This is consistent with the timing of theincrease we report, which is significant immediately followingcue offset, although there is a gradual but not significant increaseto that point throughout tone presentation that would not beexplained by a relief signal (Fig. 3b,h). Alternatively, the enhancedDA could represent a prediction error signal, independent of anyhedonic experiences, in that rats are expecting to receive a foot-shock following CS presentation but do not receive it. Althoughelectrophysiological correlates of prediction errors suggest thatthis should have a more precise temporal relationship to the tone,the diffuse nature may be explained by internal timing errors dueto the long duration of the tone and the relatively few condition-ing trials. It is perhaps more likely that when cues increase [DA]regardless of their valence, the increased DA transmission codesgeneral motivational salience, which may involve either the fearthat the cue predicts or the lack of expected footshock animalsexperience (Faure et al., 2008; Bromberg-Martin et al., 2010b;Richard and Berridge, 2011). This is consistent with previousstudies showing that increasing DA transmission within the NAcshell increases motivational responding (Ito et al., 2000; Wyvelland Berridge, 2001; Parkinson et al., 2002; Berridge, 2007).

Presentation of the aversive cue increased [DA] within theNAc shell by increasing the amplitude of phasic DA release eventssuggesting that [DA] is increased by an enhanced magnitude inthe amount of DA released per event (Aragona et al., 2009; Jones

et al., 2010). This may indicate an increase in DA neurons partic-ipating in a release event (Hyland et al., 2002). It has been shownthat aversive stimuli often drive enhanced excitation of VTA DAneurons at stimulus offset, which may contribute to the increasewe report here (Brischoux et al., 2009; Wang and Tsien, 2011).Moreover, recent work suggests that stressors potentiate shell-projecting DA neurons and increase their burst firing, which sug-gests that these neurons may be recruited during an aversivebehavioral state (Valenti et al., 2011). Further, it has been pro-posed that this modulation of DA neuron excitability is mediatedby the ventral hippocampus, a structure critical for fear-associated learning and memory (Adhikari et al., 2010; Orsini etal., 2011). Future studies will need to determine if and how thiscircuit is active during aversive cue presentations, and whetherpresentation of an aversive stimulus increases phasic transmis-sion in other brain regions such as the prefrontal cortex (Lammelet al., 2011).

ConclusionUnderstanding how aversive information modulates DA signal-ing within the NAc provides a window into how the brain per-ceives and processes motivational information. The presentstudy demonstrates the importance of phasic DA signaling withindistinct mesolimbic circuits for processing aversive stimuli.Moreover, this study uses real-time neurochemical measures infreely moving animals to address a long-standing controversyregarding how fear cues alter dopamine transmission in the NAc.We demonstrate that DA transmission does not reflect aversivecues by a simple increase or decrease, but rather the nature oftransmission depends on the specific mesolimbic pathway fromwhich the neurochemical signaling is occurring.

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