feedback control of mesolimbic somatodendritic dopamine release in rat brain

Feedback Control of Mesolimbic Somatodendritic DopamineRelease in Rat Brain

Shafiqur Rahman and William J. McBride

Department of Psychiatry, Institute of Psychiatric Research, Indiana University School of Medicine, Indianapolis, Indiana, U.S.A.

Abstract: The objective of this study was to examine therole of dopamine (DA) receptors in the nucleus accum-bens (ACB) in controlling feedback regulation of me-solimbic somatodendritic DA release in the ventral teg-mental area (VTA) of Wistar rats using ipsilateral dual-probe in vivo microdialysis. Perfusion of the ACB for 60min with the DA uptake inhibitor GBR-12909 (10–1,000mM ) or nomifensine (10–1,000 mM ) dose-dependentlyincreased the extracellular levels of DA in ACB and con-comitantly reduced the extracellular levels of DA in theVTA. Coperfusion of 100 mM nomifensine with either 100mM SCH-23390 (SCH), a D1 antagonist, or 100 mMsulpiride (SUL), a D2 receptor antagonist, produced ei-ther an additive (for SCH) or a synergistic (for SUL) ele-vation in the extracellular levels of DA in the ACB,whereas the reduction in the extracellular levels of DA inthe VTA produced by nomifensine alone was completelyprevented by addition of either antagonist. Application of100 mM SCH or SUL alone through the microdialysisprobe in the ACB increased the extracellular levels of DAin the ACB, whereas the extracellular levels of DA in theVTA remained unchanged. Overall, the results suggestthat (a) increasing the synaptic levels of DA in the ACBactivates a long-loop negative feedback pathway to theVTA involving both D1 and D2 postsynaptic receptorsand (b) terminal DA release within the ACB is regulateddirectly by D2 autoreceptors and may be indirectly reg-ulated by D1 receptors, possibly on interneurons and/orthrough postsynaptic inhibition of the negative feedbackloop. Key Words: Ventral tegmental area—Nucleus ac-cumbens—Dopamine receptors—Somatodendritic do-pamine release—Negative feedback regulation—In vivomicrodialysis.J. Neurochem. 74, 684–692 (2000).

Mesolimbic dopamine (DA) neurons originate in theventral tegmental area (VTA) and innervate the nucleusaccumbens (ACB) and other structures (Oades and Hal-liday, 1987). These neurons have been implicated inmediating the reinforcing effects of many drugs of abuse,including ethanol (Wise and Rompre, 1989; Koob, 1992;Di Chiara, 1995; Wise, 1996). The VTA has reciprocalrelationships with regions receiving its projections (Ka-livas, 1993). The ACB is known to send negative feed-back projections to the VTA (Kalivas, 1993; Kalivas

et al., 1993a,b; Lu et al., 1997, 1998; Pierce and Kalivas,1997).

Neuroanatomical, electrophysiological, and neuro-chemical studies have demonstrated the localization anddistribution of D1 and D2 receptors in the ACB (Whiteand Wang, 1986; Allin et al., 1989; Bardo and Hammer,1991; Kalivas and Duffy, 1991; Shetreat et al., 1996;Westerink et al., 1996). DA receptors have been local-ized as postsynaptic in apposition to DA terminals withinthe ACB, as presynaptic autoreceptors located on DAterminals, and as autoreceptors located at the somatoden-dritic level of DA neurons in the VTA (for review, seeMissale et al., 1998). The activity of DA neurons isregulated by short and long feedback loops. The shortfeedback system, mediated by D2-like autoreceptors atthe somatodendritic level, has been studied previously(Kalivas and Duffy, 1991; Kohl et al., 1998).

Somatodendritic DA release within the VTA (Kalivasand Duffy, 1991) provides local negative feedback reg-ulation of DA neuronal activity (Bunney et al., 1973;Wang, 1981). Perfusion of D2 antagonists in the VTAelevated the extracellular DA levels in the ACB (West-erink et al., 1996; Kohl et al., 1998), whereas perfusionof the VTA with a D2 agonist decreased the extracellularlevels of DA in the ACB (Kohl et al., 1998).

Local perfusion of D2 antagonists through microdialy-sis probes in the striatum (Imperato and Di Chiara, 1988;Westerink and de Vries, 1989; Santiago and Westerink,1991) or prefrontal cortex (Santiago et al., 1993) ele-vated the extracellular levels of DA. These results sug-gest that blocking D2 autoreceptors on DA terminalsincreases the release of DA. In addition, as in the stria-tum (Imperato et al., 1987; Imperato and Di Chiara,1988; Moghaddam and Bunney, 1990; Shi et al., 1997),

Received July 22, 1999; revised manuscript received September 24,1999; accepted September 27, 1999.

Address correspondence and reprint requests to Dr. W. J. McBride atDepartment of Psychiatry, Institute of Psychiatric Research, IndianaUniversity School of Medicine, 791 Union Drive, Indianapolis, IN46202-4887, U.S.A. E-mail: [email protected]

Abbreviations used:ACB, nucleus accumbens; ACSF, artificialCSF; DA, dopamine; SCH, SCH-23390; SUL, sulpiride; VTA, ventraltegmental area.

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Journal of NeurochemistryLippincott Williams & Wilkins, Inc., Philadelphia© 2000 International Society for Neurochemistry

the long feedback pathway from the ACB to the VTAinvolves activation of postsynaptic DA receptors.

Overall, the evidence suggests that there is a relation-ship between somatodendritic and terminal field DArelease in the mesolimbic DA system (Kohl et al., 1998)and that somatodendritic and terminal DA release isregulated by DA D2 autoreceptors. Thus far, only onestudy has demonstrated the presence of negative feed-back regulation of somatodendritic DA release in theVTA using dual-probe microdialysis (Kohl et al., 1998).However, the mechanisms within the ACB controllingthe long-loop negative feedback circuit to the VTA areunknown. Furthermore, the role of DA receptors withinthe ACB in the control of feedback pathway to the VTAhas not been established. An experimental approach thatcould be used to study the involvement of D1 and D2receptors in the control of long-loop negative feedbackregulation of VTA DA neuronal activity is with dual-probe in vivo microdialysis (Westerink et al., 1996; Kohlet al., 1998). Therefore, the present study was undertakento examine the role of DA receptors in the ACB incontrolling negative feedback regulation of VTA DAneurons using ipsilateral dual-probe in vivo microdialy-sis. The hypothesis to be tested is that postsynaptic DAD1 and D2 receptors in the ACB are involved in activa-tion of the negative feedback loop to the VTA.

MATERIALS AND METHODS

Adult male Wistar rats (weighing 250–350 g; Harlan, Indi-anapolis, IN, U.S.A.) were used in this study. Rats were singlyhoused and maintained on a normal 12-h light–dark cycle(lights on at 0700 h) in a constant temperature- and humidity-controlled animal facility with food and water ad libitum.

The following agents were used: (a) the DA uptake inhibi-tors GBR-12909z2HCl and nomifensine maleate; (b) the D1antagonistR(1)-SCH-23390zHCl (SCH); and (c) the D2 antag-onist S(2)-sulpiride (SUL) (all from RBI, Natick, MA,U.S.A.). All agents were dissolved in the microdialysis perfu-sion fluid (see below) and perfused through the microdialysisprobe in the ACB.

Rats were anesthetized under 1–2% halothane and placed inthe stereotaxic apparatus (David Kopf Instruments, Tujunga,CA, U.S.A.); the skull was exposed, and small holes weredrilled to insert guide cannulas. Rats were maintained on a37°C heating pad throughout the course of surgery. Two mi-crodialysis guide cannulas (18 gauge; Plastics One, Roanoke,VA, U.S.A.) were implanted ipsilaterally in the ACB and VTAaccording to the atlas of Paxinos and Watson (1986). Theywere implanted at a 10° angle from the midline using thefollowing coordinates relative to bregma with the incisor barset at23.4 mm: AP11.7 mm, L12.4 mm, and D/V26.3 mmfor the ACB; AP25.0 mm, L12.0 mm, and D/V27.4 mm forthe VTA. The guide cannulas were slowly (1 mm/min) insertedinto position; three stainless steel screws were placed in theskull to secure the guides, and the guides were fixed in placewith cranioplastic cement (Plastics One). Two stainless steeldummy probes cut to extend to the tip of the guide cannulaswere inserted to maintain patency. Rats were allowed to re-cover for 5–6 days in their home cages following surgery,during which they were allowed free access to food and water.All animal procedures were approved by the Institutional Re-

view Committee and are in accordance with the National In-stitutes of HealthGuidelines for the Care and Use of Labora-tory Animals.

The loop-style probes were made with dialysis tubing heatshrunk into PE-10 polyethylene tubing that were fused toPE-20 tubing. They were made as previously described (Perryand Fuller, 1992; Kohl et al., 1998). The length of the probe tipwas 2 mm for the ACB and 1.5 mm for the VTA; the totallength of the dialysis membrane was 4 and 3 mm, respectively.The loop was oriented in a rostrocaudal direction and extended;500mm. The outside diameter of the dialysis membrane was220 mm. The loop-style probes were used instead of the con-centric probes because they provided consistent and higherbasal levels of DA and sampled a large proportion of the targetarea.

On postsurgery day 5, the rats were transferred to the Plexi-glas chambers (253 44 3 38 cm, width3 length3 height),used during microdialysis, for daily handling and habituation tothe chambers. On postsurgery day 6, the rats were brieflyanesthetized with halothane, and the two loop-style probeswere inserted through the guides and cemented into place. Thefollowing day (on day 7 postsurgery), rats were placed in thePlexiglas chambers. Experiments were performed in freelymoving animals. Food and water were not available duringmicrodialysis. The inputs of the dialysis probes were connectedto a syringe pump (Harvard Instruments, South Natick, MA,U.S.A.) that delivered artificial CSF (ACSF) to the probe at arate of 0.6ml/min. The ACSF (composed of 145 mM NaCl, 2.7mM KCl, 1.0 mM MgCl2, and 1.2 mM CaCl2, pH adjusted to7.4 6 0.2 with 2 mM Na2HPO4) was filtered through a sterilefilter (pore size, 0.2mm). The ACSF was perfused for 60–90min before any baseline samples were collected. Baseline sam-ples were collected every 20 min for an additional 100 minbefore introducing any agent. Stable baseline values for theextracellular levels of DA in ACB and VTA usually occurredwithin 60 min, as previously reported (Kohl et al., 1998).Samples were collected in 0.5-ml polyethylene tubes contain-ing 3 ml of 0.05M HClO3 and were either analyzed directly orimmediately frozen on dry ice and stored at270°C untilanalysis. Frozen samples showed no sign of degradation for upto 1 month.

The DA uptake inhibitors GBR-12909 and nomifensinewere perfused through the ACB probe for 60 min to determinethe changes of extracellular DA levels in ACB and VTA. Anuptake inhibitor with SCH or SUL was perfused togetherthrough the ACB probe. Each rat was exposed to only oneconcentration of uptake inhibitor. Samples were collected be-fore, during, and 80 min after perfusion.

At the end of the experiment, 1% bromophenol blue solutionwas perfused through the probes to verify the placements. Ratswere then overdosed with CO2 and decapitated, and the brainswere removed. Brains were then stored at270°C; frozen40-mm coronal sections were prepared and stained with cresylviolet dye for verification of the probe tips. Probe placementswere evaluated according to the atlas of Paxinos and Watson(1986). Only data from animals with probe placements in boththe ACB and VTA were used.

Samples were analyzed by a microbore HPLC apparatuswith an electrochemical detection system as described (Bareet al., 1998) to determine DA levels in each sample. In brief,chromatography was performed using a model 2350 pump(ISCO, Lincoln, NE, U.S.A.) with a BAS SepStik microboreanalytical column (1.03 100 mm; 3-mm particle size Spheri-sorb C18 stationary phase) connected to a BAS custom injec-

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685DOPAMINE RELEASE IN THE VTA

tion valve and a Rheodyne 5.0-ml injection loop mounted in aBAS Unijet model CC-6 cabinet (Bioanalytical Systems, WestLafayette, IN, U.S.A.). A pulse damper (Scientific Systems,State College, PA, U.S.A.) and chromatography column (Wa-ters Spherisorb 5-mm particle size ODS-2, 33 100 mm;Keystone Scientific, Bellefonte, PA, U.S.A.) were installedin-line between the pump and injection valve to further reducepump pulsations and to increase system back pressure, respec-tively. The mobile phase was composed of 100 mM sodiumacetate, 0.5 mM EDTA, 5 mM sodium octanesulfonic acid, 10mM NaCl, and 6% acetonitrile (pH 4.0 adjusted with glacialacetic acid). The mobile phase was briefly bubbled with heliumto deoxygenate it. The column was maintained at room tem-perature, and the flow rate was 75ml/min. DA was detectedwith a BAS Unijet radial-flow detector cell with a 6-mm-diameter glassy carbon electrode (Bioanalytical Systems) cou-pled to a model 400 amperometric detector via an external cellcable (EG&G Princeton Applied Research, Princeton, NJ,U.S.A.). The applied potential was set at1450 mV with asensitivity setting of 0.5 nA/V. The use of the Unijet reference

electrode required an applied potential setting that was 100 mVless than the equivalent potential setting for a standard Ag/AgCl reference electrode. The output of the detector was sentto a Chromoperfect (Justice Innovations, Palo Alto, CA,U.S.A.) chromatography data analysis system. The lower limitof sensitivity for DA was;0.2 fmol injected onto the column.

Values were not corrected for probe recovery efficiency,which is ;15% and in good agreement with published values(Perry and Fuller, 1992). To minimize rat-to-rat variability,data for individual experiments were normalized and expressedas percent change from baseline values. Percent baseline levelsfor each experiment were calculated as treatment/control3 100. The average concentration of three stable samplesbefore perfusion with one of the DA agents (,10% variation)was considered the control and was defined as 100%. Data wereanalyzed using the statistical program Sigmastat. Normalizeddata were analyzed by one-way ANOVA followed by Dun-nett’s multiple comparison when appropriate. The significancelevel was set atp , 0.05. Time course changes were analyzed

FIG. 1. Representative locations of microdialysis probe placements in the ACB (left panels) and VTA (right panels). Overlappingplacements are not shown; therefore, the complete quantitative distribution of the placements is not indicated here. Numbers in the rightindicate the distance (in mm) from bregma (Paxinos and Watson, 1986). Black lines correspond to the location of the active membranearea of the microdialysis probes.


686 S. RAHMAN AND W. J. MCBRIDE

with a repeated-measures ANOVA without transformation ofthe data.

RESULTS

Only data from animals that had both probes correctlyimplanted in the ACB and VTA were included in thisstudy. Most (;80%) of the animals that had undergonesurgery had both probes implanted in the ACB and VTA.The loop-style probe is oriented along the anterioposte-rior axis of the ACB and VTA, and therefore a majorportion of each region is perfused. Figure 1 shows rep-resentative probe placements in the ACB and VTA; itdoes not show overlapping probe placements and is nota quantitative representation of the distribution of probeplacements. Within the ACB, almost all of the probesperfused both the core and shell to varying degrees, withsome placements mostly in the shell and some place-ments mostly in the core. A few probes did have a smallportion (,10%) of their active membrane in the striatum,and some probes had tips close to the olfactory tubercle.Because such a small portion of the active member isexposed to tissue outside the ACB, it is likely that theDA collected in the dialysis samples is mainly from thecore and shell combined. Within the VTA, a significantportion of the active membrane was located dorsal to theVTA, and no probes were located in the substantia nigra.Our previous unpublished data indicated that extracellu-lar levels of DA were not detected unless a significant

portion of the probe was located within the VTA, sug-gesting that the DA detected in the microdialysis samplesoriginated from the VTA. Contribution of DA diffusingfrom the substantia nigra to the VTA probe is not likelyto be significant because of the presence of active uptakesystems.

To examine the effect of DA uptake inhibitors onextracellular DA levels, GBR-12909 and nomifensinewere perfused for 60 min through the microdialysisprobe in the ACB. Local ACB perfusion of 10, 100, and1,000 mM GBR-12909 dose-dependently increased theextracellular levels of DA in the ACB to;150, 380, and630% of basal, respectively, and at the two highestconcentrations concomitantly reduced the extracellularlevels of DA in the VTA;50% (Fig. 2). Similarly, localACB perfusion of 10, 100, and 1,000mM nomifensineproduced dose-related increases in the extracellular lev-els of DA in the ACB to 220, 330, and 1,400% of basal,respectively, and at the two highest concentrations con-comitantly reduced the extracellular levels of DA in theVTA ;50% (Fig. 3). The small reduction in the VTAwith 10 mM nomifensine was not statistically significant.The reduction in extracellular DA levels in the VTA wasnot dose-dependent with either uptake inhibitor andreached a maximum of;50% (Figs. 2 and 3). The timecourse changes in extracellular DA levels in the ACBand VTA were nearly mirror images of each other, andboth gradually returned toward baseline when the perfu-

FIG. 2. Effects of perfusion of GBR-12909 in the ACB on extra-cellular levels of DA in the ACB (top panel) and VTA (bottompanel). GBR-12909 (0, 10, 100, or 1,000 mM ) was perfused asdescribed in Materials and Methods. After establishment of astable baseline, GBR-12909 was perfused for 60 min. Data aremean 6 SEM (bars) values of five to seven animals. One-wayANOVA revealed a significant dose effect for DA: F(3,19)5 23.03, p , 0.001 for ACB and F(3,19) 5 78.08, p , 0.001 forVTA. *p , 0.001 for difference from basal with Dunnett’s posthoc test. The basal extracellular levels of DA in the ACB and VTAwere 38 6 4 and 29 6 2 fmol/20 min, respectively.

FIG. 3. Effects of perfusion of nomifensine in the ACB on extra-cellular levels of DA in the ACB (top panel) and VTA (bottompanel). Nomifensine (0, 10, 100, or 1,000 mM ) was perfused asdescribed in Materials and Methods. After establishment of astable baseline, nomifensine was perfused for 60 min. Data aremean 6 SEM (bars) values of four to six animals. One-wayANOVA revealed a significant dose effect for DA: F(3,15)5 15.99, p , 0.001 for ACB and F(3,15) 5 13.94, p , 0.001 forVTA. *p , 0.05, **p , 0.001 for difference from basal withDunnett’s post hoc test. The basal extracellular levels of DA inthe ACB and VTA were 29 6 5 and 24 6 2 fmol/20 min, respec-tively.



sion medium was switched to ACSF (Fig. 4). Localperfusion of uptake inhibitors at the above concentra-tions did not produce any overt signs of behavioralactivation.

To determine the role of DA receptors in the ACBfollowing increased synaptic levels of DA induced byuptake inhibitors, a D1 (SCH) or D2 (SUL) antagonistwas coperfused via the microdialysis probe. Coperfusionof 100mM nomifensine with either 100mM SCH or 100mM SUL produced an additional increase in the extra-cellular levels of DA in the ACB, with increases from400% basal with nomifensine alone to 600 and 1,200%baseline with addition of SCH and SUL, respectively(Fig. 5), although the additional increase with SCH wasnot statistically significant. However, despite the addi-tional elevation in the synaptic levels of DA in the ACB,the reduction in the extracellular levels of DA in theVTA produced by nomifensine alone was completelyprevented by either antagonist (Fig. 5).

Local perfusion of 100mM, but not 1 or 10mM,SCH alone into the ACB significantly increased theextracellular levels of DA in the ACB to 175% ofbasal, whereas the extracellular levels of DA remainunchanged in the VTA at any concentration (Fig. 6).Similarly, local application of 10 and 100mM SULthrough the microdialysis probe in the ACB produceddose-related increases in the extracellular levels of DA

in the ACB to 140 and 200% of basal, respectively,whereas DA levels in the VTA remain unaltered at anyconcentration (Fig. 7). However, the small increase inDA levels in the ACB with 10mM SUL was notstatistically significant.

DISCUSSION

The major findings of this study suggest that (a) thereis negative feedback regulation from the ACB to theVTA (Figs. 2–4), (b) D1 and D2 receptors within theACB are essential in mediating the activation of thisnegative feedback pathway to the VTA (Fig. 5), and (c)terminal DA release in the ACB is regulated directly byD2 autoreceptors (Fig. 7) and indirectly by D1 receptors(Fig. 6).

Local perfusion of DA uptake inhibitors through thedialysis probe in the ACB increased the extracellular levelsof DA in the ACB in a dose-related manner (Figs. 2 and 3).These data are consistent with previous findings using mi-crodialysis probes in the ACB (Kalivas and Duffy, 1991; Liet al., 1996; Reith et al., 1997; Kohl et al., 1998) or otherDA target regions (Carboni et al., 1989; Nomikos et al.,1990; Santiago and Westerink, 1991; Nakachi et al., 1995;Engberg et al., 1997). GBR-12909 and nomifensine pro-duced similar effects on synaptic DA levels in the range10–100mM (Figs. 2 and 3). However, the highest concen-tration of nomifensine used in this study elevated extracel-lular levels of DA in the ACB twice the amount producedby GBR-12909. The underlying mechanism for this actionremains unclear, although it is possible that the greaterincrease of DA levels with 1,000mM nomifensine may be

FIG. 4. Time course of the effects of local ACB perfusion for 60min with 100 mM nomifensine on extracellular levels of DA in theACB (top panel) and VTA (bottom panel). Nomifensine wasperfused starting at the zero time point. Data are mean 6 SEM(bars) values of three animals. Data (without transformation)were analyzed by one-way repeated-measures ANOVA withDunnett’s post hoc test for each region separately. Significantdifferences were F(6,12) 5 3.76, *p , 0.05 for ACB and F(6,12)5 4.83, *p , 0.05 for VTA. The basal extracellular levels of DA inthe ACB and VTA were 43 6 14 and 26 6 2 fmol/20 min,respectively.

FIG. 5. Effects of coperfusion of nomifensine (Nom; 100 mM )and SCH (100 mM ) or SUL (100 mM ) in the ACB on extracellularDA levels in the ACB (top panel) and VTA (bottom panel). Dataare mean 6 SEM (bars) values of four or five animals. One-wayANOVA revealed a significant dose effect for DA: F(2,11) 5 4.88,p , 0.05 for ACB and F(2,9) 5 23.18, p , 0.001 for VTA. *p, 0.001 for difference from Nom with Dunnett’s post hoc test.The basal extracellular levels of DA in the ACB and VTA were 416 12 and 26 6 6 fmol/20 min, respectively.



due to nonselective actions on other systems regulating DArelease, as demonstrated in superfused rat striatal slices(Herdon et al., 1987).

Concurrent with the increase in extracellular levelsof DA in the ACB with the two highest concentrationsof the uptake inhibitors there was a 50% decrease inthe extracellular levels of DA in the VTA (Figs. 2 and3). The 400% increase in extracellular DA levels in theACB and the 50% reduction in DA levels in the VTAwith the 100 mM concentration of GBR-12909 aresimilar to results previously reported (Kohl et al.,1998). The reduction in extracellular levels of DA inthe VTA may reflect a reduction in somatodendriticDA release produced by activation of a long-loopnegative feedback pathway. The time course forchanges in synaptic DA levels in the ACB is in goodagreement with the time course for changes in soma-todendritic release in the VTA (Fig. 4), supporting theidea of an activation of a negative feedback pathwayfrom the ACB to the VTA. However, the reduction ofsomatodendritic DA release in the VTA was similar atthe two highest doses of the uptake inhibitors eventhough there were dose-related increases in extracel-lular DA levels in the ACB (Figs. 2 and 3). The mostlikely reasons for this finding are that (a) the synapticlevels of DA are sufficiently high at the intermediatedose to activate the maximal number of receptorsregulating the long-loop feedback system and (b) notall neurons within the VTA are regulated by negativefeedback from the ACB.

Coperfusion of the ACB with nomifensine and eitherSCH or SUL completely prevented the reduction in theextracellular levels of DA in the VTA (Fig. 5), suggest-ing that both D1 and D2 receptors are required to activatethe negative feedback loop to the VTA. Binding and insitu hybridization studies provide evidence indicatinglocalization of D1 and D2 receptors within the ACB tomedium-size spiny GABAergic projection neurons(Bardo and Hammer, 1991; Shetreat et al., 1996; forreview, see Missale et al., 1998). However, within theACB, D1 and D2 receptors appear to be mainly segre-gated with only a small population of neurons coexpress-ing both receptors, and these appeared to be mainlyinterneurons (Meredith et al., 1993; Pennartz et al., 1994;for review, see Missale et al., 1998). Findings fromelectrophysiological studies in the ACB are consistentwith a postsynaptic localization for D1 and D2 receptors(White and Wang, 1986). Moreover, electrophysiologi-cal (White, 1987; Clark and White, 1987; Wachtel et al.,1989) and behavioral (for review, see Clark and White,1987) findings indicate that D1 receptor stimulation isneeded to produce many postsynaptic D2 receptor-me-diated effects in the ACB. The present findings arecompatible with these results and may indicate that D1receptor stimulation is required to enable postsynapticD2 receptor-mediated responses to activate the ACBnegative feedback system.

It is possible that DA uptake inhibitors, by increasingthe availability of DA in the ACB, activate the GABAer-gic feedback pathway from the ACB to the VTA (Walaas

FIG. 6. Effects of perfusion of SCH in the ACB on extracellularlevels of DA in the ACB (top panel) and VTA (bottom panel).After establishment of a stable baseline, SCH (0, 1, 10, or 100mM ) was perfused for 60 min as described in Materials andMethods. Data are mean 6 SEM (bars) values of five to sevenanimals. One-way ANOVA revealed a significant dose effect forDA: F(3,17) 5 3.69, p , 0.05 for ACB. *p , 0.001 for differencefrom basal with Dunnett’s post hoc test. The basal extracellularlevels of DA in the ACB and VTA were 31 6 5 and 24 6 5 fmol/20min, respectively.

FIG. 7. Effects of perfusion of SUL in the ACB on extracellularlevels of DA in the ACB (top panel) and VTA (bottom panel).After establishment of a stable baseline, SUL (0, 1, 10, or 100mM ) was perfused for 60 min as described in Materials andMethods. Data are mean 6 SEM (bars) values of five to sevenanimals. One-way ANOVA revealed a significant dose effect forDA: F(3,16) 5 12.73, p , 0.001 for ACB. *p , 0.001 for differ-ence from basal with Dunnett’s post hoc test. The basal extra-cellular levels of DA in the ACB and VTA were 34 6 5 and 26 6 5fmol/20 min, respectively.



and Fonnum, 1980; Kalivas et al., 1993a). However, thelarge majority of neurons projecting from the ACB to theVTA expresses mRNA for the D1 receptor with only asmall percentage expressing mRNA for the D2 receptor(Lu et al., 1998). Therefore, it is difficult to reconcile thepresent findings in terms of an interaction of D1 and D2receptors directly on neurons that project from the ACBto the VTA. The action of SCH in blocking the reductionin extracellular DA levels in the VTA, produced bynomifensine, may occur through inhibition of D1 recep-tors on output neurons projecting to the VTA. However,a similar effect by the D2 antagonist on the extracellularDA levels in the VTA would have to occur through adifferent mechanism.

Expression of mRNA for the D2 receptor has beenfound in cholinergic neurons within the ACB (Mac-Lennan et al., 1994) and in neurons projecting fromthe ACB to the ventral pallidum (Lu et al., 1998). Inaddition, neurons projecting from the ACB to theventral pallidum also express mRNA for the D1 re-ceptor (Lu et al., 1998). Therefore, the involvement ofD2 receptors within the ACB, in mediating negativefeedback inhibition of VTA DA neurons, could occurthrough activation of D2 receptors on cholinergic in-terneurons (Meredith et al., 1993; MacLennan et al.,1994), which control the activity of the ACB outputneurons. A similar mechanism could be envisionedfor D2 receptors on excitatory amino acid afferents(Meredith et al., 1993; Pennartz et al., 1994; Pierceand Kalivas, 1997) to ACB output neurons. In bothcases, activation of D2 receptors produces inhibi-tion of these excitatory inputs to the output neuronsand shifts the balance of control over the outputneurons toward inhibition. Therefore, activation ofD1 receptors could produce inhibitory effects directlyon the output neurons, whereas activation of D2 re-ceptors could produce inhibitory effects indirectly onthe output neurons via inhibition of direct excitatoryinputs.

Another possibility is that D1 and D2 receptors colo-calized on excitatory cholinergic interneurons and/or ex-citatory amino acid inputs within the ACB may be in-volved in activation of the negative feedback system. Inthis case, D1 receptor stimulation may be necessary toenable D2 receptor-mediated inhibition. In addition, amajor GABAergic output from the ACB goes to theventral pallidum (for review, see Kalivas et al., 1993b),and these output neurons express mRNAs for D1 and D2receptors (Lu et al., 1998). Therefore, because the ventralpallidum also has a reciprocal GABA feedback connec-tion to the VTA (for review, see Kalivas et al., 1993b), itis possible that the reduction in VTA DA release pro-duced by elevated synaptic DA levels in the ACB couldbe mediated through the ACB output projections to theventral pallidum.

If inhibition of spiny GABA output neurons within theACB to the VTA is involved in mediating long-loopfeedback, then this process would involve a disinhibition

mechanism, i.e., release of VTA GABA interneuronsfrom tonic inhibition (for review, see Kalivas, 1993). Adisinhibition process could also account for feedbackinhibition mediated through the ventral pallidum. In thiscase, inhibition of GABA output neurons from the ACBto the ventral pallidum disinhibits the ventral pallidumGABA output neurons, which project to the VTA andcan directly inhibit DA neuronal activity (for review, seeKalivas, 1993).

Local perfusion with 100mM SCH alone signifi-cantly increased the extracellular levels of DA in theACB to 175% of basal values and did not alter theVTA dialysis concentrations of DA (Fig. 6). The ob-servation that SCH alone increased the extracellularlevels of DA in the ACB is in agreement with previousmicrodialysis studies (Imperato and Di Chiara, 1988;Santiago et al., 1993). SCH might stimulate DA re-lease in the ACB by blocking D1 receptors on theGABAergic output neurons mediating negative feed-back regulation to the VTA and/or blocking D1-me-diated inhibition of excitatory interneurons, whichmay be involved in local or long-loop regulation ofDA release.

D2 receptors suppress the functional activity of DAneurons at various critical sites, i.e., impulse generation,DA synthesis, and DA release (Wolf and Roth, 1987).SUL alone at the highest concentration increased theextracellular levels of DA in the ACB to 200% of base-line, whereas DA levels in the VTA remain unchanged(Fig. 7). The increased extracellular levels of DA withSUL suggests that blocking D2 nerve terminal autore-ceptors is the likely mechanism underlying this effect.This finding is also in conformity with the results ofprevious microdialysis studies (Imperato and Di Chiara,1988; Westerink and de Vries, 1989; Santiago et al.,1993).

Local application of SUL along with nomifensineproduced a synergistic effect on the extracellular lev-els of DA in the ACB, whereas coinfusion of SCHwith nomifensine did not have this effect (Fig. 5). Thesynergistic elevation of extracellular DA levels in theACB produced by the combination of SUL and nomi-fensine is in agreement with a previous report indicat-ing that coinfusion of these agents potentiated extra-cellular levels of DA in the striatum (Santiago andWesterink, 1991). This synergistic effect on extracel-lular levels of DA in the ACB may result from simul-taneously blocking three mechanisms controllingsynaptic DA levels, i.e., reuptake, D2 autoreceptors,and D2 regulation of the long-loop negative feedbacksystem.

In summary, the results of the present study indicatethat increasing synaptic levels of DA in the ACB termi-nal region can activate long-loop negative feedback con-trol of VTA DA neurons. Moreover, activation of thisnegative feedback system requires both D1 and D2 re-ceptors.



Acknowledgment: This work was supported in part by U.S.Public Health Service grant AA10721. The skillful technicalassistance of Miranda Henry is gratefully acknowledged.

REFERENCES

Allin R., Russell V., Lamm M., and Taljaard J. (1989) Regionaldistribution of dopamine D1 and D2 receptors in the nucleusaccumbens of the rat.Brain Res.501,389–391.

Bare D. J., McKinzie J. H., and McBride W. J. (1998) Development ofrapid tolerance to ethanol stimulated serotonin release in theventral hippocampus.Alcohol. Clin. Exp. Res.22, 1272–1276.

Bardo M. T. and Hammer R. P. Jr. (1991) Autoradiographic localiza-tion of dopamine D1 and D2 receptors in rat nucleus accumbens:resistance to differential rearing conditions.Neuroscience45,281–290.

Bunney B. S., Walters J. R., Roth R. H., and Aghajanian G. K. (1973)Dopaminergic neurons: effect of antipsychotic drugs and amphet-amine on single cell activity.J. Pharmacol. Exp. Ther.185,560–571.

Carboni E., Imperato A., Perezzani L., and Di Chiara G. (1989)Amphetamine, cocaine, phencyclidine and nomifensine increaseextracellular dopamine concentrations preferentially in the nu-cleus accumbens of freely moving rats.Neuroscience28, 653–661.

Clark D. and White F. J. (1987) D1 dopamine receptor—the searchfor a function: a critical evaluation of the D1/D2 dopaminereceptor classification and its functional implications.Synapse1, 347–388.

Di Chiara G. (1995) The role of dopamine in drug abuse viewed fromthe perspective of its role in motivation.Drug Alcohol Depend38,95–137.

Engberg G., Elverfors A., Jonson J., and Nissbrandt H. (1997) Inhibi-tion of dopamine reuptake: significance for nigral dopamine neu-ron activity.Synapse25, 215–226.

Herdon H., Strupish J., and Nahorski S. R. (1987) Endogenous dopa-mine release from rat striatal slices and its regulation by D-2autoreceptors: effects of uptake inhibitors and synthesis inhibition.Eur. J. Pharmacol.138,69–76.

Imperato A. and Di Chiara G. (1988) Effects of locally applied D-1 andD-2 receptor agonists and antagonists studied with brain dialysis.Eur. J. Pharmacol.156,385–393.

Imperato A., Mulas A., and Di Chiara G. (1987) The D-1 antagonistSCH 23390 stimulates while the D-1 agonists SKF 38393 fails toaffect dopamine release in the dorsal caudate of freely movingrats.Eur. J. Pharmacol.142,177–181.

Kalivas P. W. (1993) Neurotransmitter regulation of dopamine neuronsin the ventral tegmental area.Brain Res. Rev.18, 75–113.

Kalivas P. W. and Duffy P. (1991) A comparison of axonal andsomatodendritic dopamine release using in vivo dialysis.J. Neu-rochem.56, 961–967.

Kalivas P. W., Churchill L., and Kiltenick M. A. (1993a) GABA andenkephalin projections from the nucleus accumbens and ventralpallidum to VTA. Neuroscience57, 1047–1060.

Kalivas P. W., Churchill L., and Klitenick M. A. (1993b) The circuitrymediating the translation of motivational stimuli into adaptivemotor responses, inLimbic Motor Circuits and Neuropsychiatry(Kalivas P. W. and Barnes C. D., eds), pp. 237–288. CRC Press,Boca Raton, Florida.

Kohl R. R., Katner J. S., Chernet E., and McBride W. J. (1998) Ethanoland negative feedback regulation of mesolimbic dopamine releasein rats.Psychopharmacology (Berl.)139,79–85.

Koob G. F. (1992) Drugs of abuse: anatomy, pharmacology andfunction of reward pathways.Trends Pharmacol. Sci.13, 177–184.

Li M.-Y., Yan Q.-S., Coffey L. L., and Reith M. E. A. (1996) Extra-cellular dopamine, norepinephrine, and serotonin in the nucleusaccumbens of freely moving rats during intracerebral dialysis withcocaine and other monoamine uptake blockers.J. Neurochem.66,559–568.

Lu X.-Y., Churchill L., and Kalivas P. W. (1997) Expression of D1receptor mRNA in projections from the forebrain to the ventraltegmental area.Synapse25, 205–214.

Lu X.-Y., Ghasemzadeh M. B., and Kalivas P. W. (1998) Expression ofD1 receptor, D2 receptor, substance P and enkephalin messengerRNAs in neurons projecting from the nucleus accumbens.Neuro-science82, 767–780.

MacLennan A. J., Lee N., Vincent S. R., and Walker D. W. (1994) D2dopamine receptor mRNA distribution in cholinergic and soma-tostatinergic cells of the rat caudate-putamen and nucleus accum-bens.Neurosci. Lett.180,214–218.

Meredith G. E., Pennartz C. M. A., and Groenewegen H. J. (1993) Thecellular framework for chemical signalling in the nucleus accum-bens.Prog. Brain Res.9, 3–24.

Missale C., Nash S. R., Robinson S. W., Jaber M., and Caron M. G.(1998) Dopamine receptors: from structure to function.Physiol.Rev.78, 189–225.

Moghaddam B. and Bunney B. S. (1990) Acute effects of typical andatypical antipsychotic drugs on the release of dopamine fromprefrontal cortex, nucleus accumbens, and striatum of the rat: an invivo microdialysis study.J. Neurochem.54, 1755–1760.

Nakachi N., Kiuchi Y., Inagaki M., Inazu M., Yamazaki Y., andOguchi K. (1995) Effects of various dopamine uptake inhibitorson striatal extracellular dopamine levels and behaviors in rats.Eur. J. Pharmacol.281,195–203.

Nomikos G. G., Damsma G., Wenkstern D., and Fibiger H. C. (1990)In vivo characterization of locally applied dopamine uptake in-hibitors by striatal microdialysis.Synapse6, 106–112.

Oades R. D. and Halliday G. M. (1987) Ventral tegmental (A10)system: neurobiology. I. Anatomy and connectivity.Brain Res.Rev.12, 117–165.

Paxinos G. and Watson C. (1986)The Rat Brain in Stereotaxic Coor-dinates.Academic Press, New York.

Pennartz C. M. A., Groenwegen H. J., and Lopes da Silva F. H.(1994) The nucleus accumbens as a complex of functionallydistinct neuronal ensembles: an integration of behavioural,electrophysiological and anatomical data.Prog. Neurobiol.42,719 –761.

Perry K. W. and Fuller R. W. (1992) Effect of fluoxetine on serotoninand dopamine concentration in microdialysis fluid from rat stria-tum. Life Sci.50, 1683–1690.

Pierce R. C. and Kalivas P. W. (1997) A circuitry model of theexpression of behavioral sensitization to amphetamine-like psy-chostimulants.Brain Res. Rev.25, 192–216.

Reith M. E. A., Li M.-Y., and Yan Q.-S. (1997) Extracellulardopamine, norepinephrine, and serotonin in the ventral tegmen-tal area and nucleus accumbens of freely moving rats duringintracerebral dialysis following systemic administration of co-caine and other uptake blockers.Psychopharmacology (Berl.)134, 309 –317.

Santiago M. and Westerink B. H. C. (1991) Characterization andpharmacological responsiveness of dopamine release recorded bymicrodialysis in the substantia nigra of conscious rats.J. Neuro-chem.57, 738–747.

Santiago M., Machado A., and Cano J. (1993) Regulation of prefrontalcortical dopamine release by dopamine receptor agonists andantagonists.Eur. J. Pharmacol.239,83–91.

Shetreat M. E., Lin L., Wong A. C., and Rayport S. (1996) Visualiza-tion of D1 dopamine receptors on living nucleus accumbensneurons and their colocalization with D2 receptors.J. Neurochem.66, 1475–1482.

Shi W.-X., Smith P. L., Pun C.-L., Millet B., and Bunney B. S. (1997)D1–D2 interaction in feedback control of midbrain dopamineneurons.J. Neurosci.15, 7988–7994.

Wachtel S. R., Hu X.-T., Galloway M. P., and White F. J. (1989)D1 dopamine receptor stimulation enables the postsynaptic, butnot autoreceptor, effects of D2 dopamine agonists in nigrostri-atal and mesoaccumbens dopamine systems.Synapse4, 327–346.

Walaas I. and Fonnum F. (1980) Biochemical evidence forg-aminobu-tyrate containing fibres from the nucleus accumbens to the sub-



stantia nigra and ventral tegmental area in the rat.Neuroscience5,63–72.

Wang R. Y. (1981) Dopaminergic neurons in the rat ventral teg-mental area. II. Evidence for autoregulation.Brain Res. Rev.3,141–152.

Westerink B. H. C. and de Vries J. B. (1989) On the mechanism ofneuroleptic induced increase in striatal dopamine release: braindialysis provides direct evidence for mediation by autoreceptorslocalized on nerve terminals.Neurosci. Lett.99, 197–202.

Westerink B. H. C., Kwint H.-F., and de Vries J. B. (1996) Thepharmacology of mesolimbic dopamine neurons: a dual-probemicrodialysis study in the ventral tegmental area and nucleusaccumbens of the rat brain.J. Neurosci.16, 2605–2611.

White F. J. (1987) D-1 dopamine receptor stimulation “enables” theinhibitory effects of D-2 agonist quinpirole on rat nucleus accum-bens neurons.Eur. J. Pharmacol.135,101–105.

White F. J. and Wang R. Y. (1986) Electrophysiological evidence forthe existence of both D-1 and D-2 dopamine receptors in the ratnucleus accumbens.J. Neurosci.6, 274–280.

Wise R. A. (1996) Addictive drugs and brain stimulation reward.Annu.Rev. Neurosci.19, 319–340.

Wise R. A. and Rompre P. P. (1989) Brain dopamine and reward.Annu. Rev. Psychol.40, 191–225.

Wolf M. E. and Roth R. H. (1987) Dopamine autoreceptors, inDopa-mine Receptors(Creese I. and Fraser C. M., eds), p. 45. Alan R.Liss, New York.



feedback control of mesolimbic somatodendritic dopamine release in rat brain

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