involvement of gaba and cholinergic receptors in the nucleus accumbens on feedback control of...

Involvement of GABA and cholinergic receptors in the nucleus

accumbens on feedback control of somatodendritic dopamine

release in the ventral tegmental area

Shafiqur Rahman1 and William J. McBride

Department of Psychiatry, Institute of Psychiatric Research, Indiana University School of Medicine, Indianapolis, Indiana, USA

Abstract

The objectives of the present study were to examine the

involvement of GABA and cholinergic receptors within the

nucleus accumbens (ACB) on feedback regulation of

somatodendritic dopamine (DA) release in the ventral teg-

mental area (VTA). Adult male Wistar rats were implanted with

ipsilateral dual guide cannulae for in vivo microdialysis stud-

ies. Activation of the feedback system was accomplished by

perfusion of the ACB with the DA uptake inhibitor GBR 12909

(GBR; 100 lM). To assess the involvement of GABA and

cholinergic receptors in regulating this feedback system,

antagonists (100 lM) for GABAA (bicuculline, BIC), GABAB

(phaclofen, PHAC), muscarinic (scopolamine, SCOP), and

nicotinic (mecamylamine, MEC) receptors were perfused

through the probe in the ACB while measuring extracellular

DA levels in the ACB and VTA. Local perfusion of the ACB

with GBR significantly increased (500% of baseline) the

extracellular levels of DA in the ACB and produced a

concomitant decrease (50% of baseline) in the extracellular

DA levels in the VTA. Perfusion of the ACB with BIC or PHAC

alone produced a 200–400% increase in the extracellular

levels of DA in the ACB but neither antagonist altered the

levels of DA in the VTA. Co-perfusion of either GABA receptor

antagonist with GBR further increased the extracellular levels

of DA in the ACB to 700–800% of baseline. However,

coperfusion with BIC completely prevented the reduction in

the extracellular levels of DA in the VTA produced by GBR

alone, whereas PHAC partially prevented the reduction. Local

perfusion of the ACB with either MEC or SCOP alone had little

effect on the extracellular levels of DA in the ACB or VTA.

Co-perfusion of either cholinergic receptor antagonist with

GBR markedly reduced the extracellular levels of DA in the

ACB and prevented the effects of GBR on reducing DA levels

in the VTA. Overall, the results of this study suggest that

terminal DA release in the ACB is under tonic GABA inhibition

mediated by GABAA (and possibly GABAB) receptors, and

tonic cholinergic excitation mediated by both muscarinic and

nicotinic receptors. Activation of GABAA (and possibly

GABAB) receptors within the ACB may be involved in the

feedback inhibition of VTA DA neurons. Cholinergic inter-

neurons may influence the negative feedback system by

regulating terminal DA release within the ACB.

Keywords: cholinergic receptors, dopamine, feedback

regulation, GABA receptors, nucleus accumbens, ventral

tegmental area.

J. Neurochem. (2002) 80, 646–654.

One of the main target areas of the mesolimbic dopamine

(DA) neurons originating in the ventral tegmental area (VTA)

is the nucleus accumbens (ACB). The ACB is known to send

negative feedback projections to the VTA (Kalivas 1993;

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

Kalivas 1997). Previous microdialysis studies indicated that

increasing the extracellular levels of DA in the ACB with

local perfusion of a DA uptake inhibitor produced a

concomitant decrease in the extracellular levels of DA in

the VTA (Kohl et al. 1998; Rahman and McBride 2000,

2001). These results suggested that activating DA receptors

in the ACB reduced somatodendritic DA release in the VTA

Received August 27, 2001; revised manuscript received November 16,

2001; accepted November 19, 2001.

Address correspondence and reprint request to Dr W. J. McBride,

Institute of Psychiatric Research, 791 Union Drive, Indianapolis, IN

46202–4887, USA. E-mail: [email protected] address: Center for Addiction and Mental Health and

Department of Psychiatry, University of Toronto, 33 Russell St, Toronto,

Ontario M5S 2S1, Canada.

Abbreviations used: ACB, nucleus accumbens; ACSF, artificial

cerebrospinal fluid; BIC, bicuculline; DA, dopamine; MEC, mecamyl-

amine; PHAC, phaclofen; SCOP, scopolamine; VTA, ventral tegmental

area.

Journal of Neurochemistry, 2002, 80, 646–654

646 Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry, 80, 646–654

via a long-loop negative feedback system. These microdial-

ysis studies also indicated that concurrent activation of both

D1- and D2-like receptors was involved in this feedback

process (Rahman and McBride 2000, 2001). However, the

mechanisms underlying the interaction of the D1- and D2-

like receptors within the ACB in mediating this feedback

process are not known.

Binding and in situ hybridization studies provide evidence

indicating the localization of D1 and D2 receptors within the

ACB to medium-size spiny GABAergic projection neurons

(Bardo and Hammer 1991; Shetreat et al. 1996; Missale

et al. 1998). Within the ACB, D1 and D2 receptors appear to

be mainly segregated with only a small population of neurons

coexpressing both receptors, and these appear to be mainly

interneurons (Jongen-Relo et al. 1995; Meredith et al. 1993;

Pennartz et al. 1994; Missale et al. 1998). The large majority

of neurons projecting from the ACB to the VTA express

mRNA for the D1 receptor with only a small percentage

expressing mRNA for the D2 receptor (Lu et al. 1998).

Therefore, a direct interaction of D1 and D2 receptors on

medium spiny GABAergic neurons projecting from the ACB

to the VTA does not appear to be a mechanism underlying

regulation of the long-loop negative feedback pathway.

Expression of mRNA for the D2 receptor has been found

in cholinergic neurons within the ACB (MacLennan et al.

1994) and in GABA projection neurons from the ACB to the

ventral pallidum (Lu et al. 1998), whereas D1 receptor

mRNA was expressed in GABAergic output neurons to the

VTA (Lu et al. 1998). An interaction of cholinergic and DA

receptors within the neostriatum has been reported (Harsing

and Zigmond 1998). Therefore, it is possible that D1–D2

interactions within the ACB on the negative feedback loop

could occur via D2 inhibition of release of acetylcholine at

inhibitory M4 receptors concurrently with D1 excitation of

GABA output neurons (Di Chiara et al. 1994). In addition,

medium spiny neurons within the ACB send collaterals to

other GABA neurons (McGinty 1999; Meredith 1999), and it

is possible that the interaction of D1 and D2 receptors on the

feedback system to the VTA may be mediated partly through

medium spiny GABA neuron collaterals (or GABA inter-

neurons) modulation of GABA output neurons projecting to

the VTA. In this case, activation of D2 receptors on one set

of GABA neurons would disinhibit the GABA output

neurons projecting to the VTA; concurrent activation of D1

receptors on these GABA output neurons would then result

in the activation of the negative feedback loop. Cholinergic

modulation of terminal DA release (Marshall et al. 1997;

Smolders et al. 1997; Wonnacott et al. 2000) within the

ACB could also be involved in regulating the long-loop

feedback pathway.

The present study was undertaken to test the hypothesis

that GABA collaterals and/or cholinergic neurons within the

ACB are involved in the long-loop negative feedback control

of VTA DA neuronal activity. To test this hypothesis, an

ipsilateral dual probe microdialysis procedure was used to

locally manipulate the DA, GABA and cholinergic systems

within the ACB while simultaneously measuring somato-

dendritic DA release in the VTA (to assess activation of the

feedback pathway) and terminal DA release in the ACB (to

assess DA synaptic activity).

Materials and methods

Adult male Wistar rats (250–350 g; Harlan Inc., Indianapolis, IN,

USA) were used in this study. Rats were singly housed and

maintained on a normal 12 h light-dark cycle (lights on 07.00) in a

constant temperature and humidity controlled animal facility with

food and water ad libitum.

The following agents were used: DA uptake inhibitor GBR

12909.2HCl (GBR); GABAA receptor antagonist (–) bicuculline

methbromide (BIC); GABAB receptor antagonist phaclofen

(PHAC); muscarinic receptor antagonist (–)-scopolamine methyl-

bromide (scopolamine; SCOP); and the nicotinic receptor antagonist

mecamylamine.HCl (MEC). All chemicals were purchased from

Research Biochemicals Inc. (Natick, MA, USA). All agents were

dissolved in the microdialysis perfusion fluid (see below) and

perfused through the microdialysis probe in the ACB.

Rats were anaesthetized with 1–2% isoflurane and placed in the

stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA);

the skull was exposed and small holes were drilled to insert guide

cannulae. Rats were maintained on a 37°C heating pad throughout

the course of surgery. Two microdialysis guide cannulae (18 gauge;

Plastics One, Roanoke, VA, USA) were implanted ipsilaterally in

the ACB and VTA according to the atlas of Paxinos and Watson

(1986). They were implanted at a 10° angle from the midline using

the following coordinates with the incisor bar set at )3.3 mm:

AP + 1.7 mm from bregma, L + 2.4 mm, and D/V )6.3 mm for the

ACB, and AP )5.0 mm from bregma, L + 2.0 mm and D/V

)7.4 mm for the VTA. The guide cannulae were slowly (1 mm/min)

inserted into position; three stainless steel screws were placed in the

skull to secure the guides, and the guides were fixed in place with

cranioplastic cement (Plastics One). Two stainless steel dummy

probes, cut to extend to the tip of the guide cannulae, were inserted

to maintain patency. Rats were allowed to recover for 5–6 days in

their home cages following surgery, during which they were allowed

free access to food and water. Animals used in these procedures

were maintained in facilities fully accredited by the Association for

the Assessment and Accreditation of Laboratory Animal Care

(AAALAC). All research protocols were approved by the institu-

tional animal care and use committee and are in accordance with the

guidelines of the Institutional Care and Use Committee of the

National Institute on Drug Abuse, NIH, and the Guide for the Care

and Use of Laboratory Animals (Institute of Laboratory Animal

Resources, Commission on Life Sciences, National Research

Council 1996).

The loop style probes were made with dialysis tubing heat shrunk

into PE-10 polyethylene tubing that was fused to PE-20 tubing.

Probes were made as previously described (Perry and Fuller 1992;

Kohl et al. 1998). The length of the probe tip was 2 mm for the

ACB and 1.5 mm for the VTA; the total length of the dialysis

membrane was 4 and 3 mm, respectively. The loop was oriented in a

Feedback regulation of the VTA 647

Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry, 80, 646–654

rostral-caudal direction and extended approximately 500 lm. The

outside diameter of the dialysis membrane was 220 lm. The loop

style probes were used instead of the concentric probes because they

provided consistent and higher basal levels of DA, and sampled a

large proportion of the target area.

On post surgery day 5, rats were transferred to the plexiglass

chambers (25 · 44 · 38 cm, W · L · H), used during microdi-

alysis, for daily handling and habituation to the chambers. They

remained in the chambers for approximately 4–5 h. On post

surgery day 6, the rats were briefly anaesthetized with isoflurane

and the two loop style probes were inserted through the guides

and cemented into place. The following day (on day 7 post

surgery), rats were placed in the plexiglass chambers. Experi-

ments were performed in freely moving animals. Food and water

were not available during microdialysis but were available at all

other times. The input of the dialysis probes were connected to a

syringe pump (Harvard Instruments, South Natick, MA, USA),

which delivered artificial cerebrospinal fluid (ACSF) to the probe

at a rate of 0.6 lL/min. The ACSF (composition in mM: NaCl,

145; KCl, 2.7; MgCl2, 1.0; CaCl2, 1.2; pH adjusted to 7.4 ± 0.2

with 2 mM Na2HPO4) was filtered through a 0.2-lm sterile filter.

The ACSF was perfused for 60–90 min prior to collecting the

baseline samples. This procedure has been shown to give stable

calcium-dependent basal extracellular levels of DA (Bowers et al.

2000; Westerink and De Vries 1988; Campbell and McBride

1995; Campbell et al. 1996; Kohl et al. 1998). Baseline samples

were collected every 20 min for an additional 60 min before

introducing any agent. Stable baseline values for the extracellular

levels of DA in the ACB and VTA usually occurred within

60 min, as previously reported (Kohl et al. 1998; Rahman and

McBride 2000, 2001). Samples were collected in 0.5 mL

polyethylene tubes containing 3 lL of 0.05 N HClO4 and were

either analyzed directly or immediately frozen on dry ice and

stored at )70°C until analysis. The entire sample was used to

ensure that the 5 lL injection loop was completely filled. Frozen

samples showed no sign of degradation for up to one month.

All agents were perfused through the ACB probe for 60 min to

determine the effects on the extracellular levels of DA in the

ACB and VTA. Separate groups were perfused with GBR alone,

antagonist alone, or the combination of GBR plus antagonist.

Each rat was exposed to only one agent or combination. Baseline

samples were collected every 20 min for 60 min before switching

to the treatment. Antagonists alone or in combination with GBR

were then perfused for 60 min before switching back to ACSF

for an additional 60 min; 20 min samples were collected

throughout. The timing of the sample collection was corrected

for the length of the exit line between the probe and the

collection tube. The concentration of GBR used in the present

study was one which produced a reliable but submaximal effect

on the extracellular levels of DA in the ACB and effectively

reduced somatodendritic DA release in the VTA (Rahman and

McBride 2000). The 100 lM concentration used for the antago-

nists was similar to concentrations used in other microdialysis

studies with DA (Rahman and McBride 2000), GABA (Westerink

et al. 1996; Ikemoto et al. 1997), and cholinergic (Blomqvist

et al. 1997; Smolders et al. 1997) antagonists. The 100 lM

concentration of BIC (Santiago and Westerink 1992; Smolders

et al. 1995; Westerink et al. 1996; Yan 1999), PHAC (Smolders

et al. 1995; Westerink et al. 1996; Gong et al. 1998), SCOP

(Rawls and McGinty 1998; Westerink et al. 1996), and MEC

(Nisell et al. 1994; Westerink et al. 1996; Blomqvist et al. 1997)

has been used in a number of microdialysis studies and has been

shown to produce reasonably selective pharmacological effects.

Although the actual tissue concentrations of the reagents are not

known, the concentrations are likely to be significantly lower than

the amount in the probe.

At the end of the experiment, 1% bromphenol blue solution

was perfused through the probes to verify the placements. Rats

were then overdosed with CO2, decapitated and the brains

removed. Brains were then stored at )70°C; frozen 40 lmcoronal sections were prepared and stained with cresyl violet dye

for verification of the probe tips. Probe placements were

evaluated according to the atlas of Paxinos and Watson (1986).

Only data from animals with probe placements in both the ACB

and VTA were used.

Samples were analyzed by microbore HPLC with an electro-

chemical detection system as described (Rahman and McBride

2000) to determine DA levels in each sample. Briefly, chroma-

tography was performed using a model 2350 pump (ISCO,

Lincoln, NE, USA) with a BAS SepStik microbore analytical

column (1.0 · 100 mm column; 3 lm Spherisorb C18 stationary

phase) connected to a BAS custom injection valve and a

Rheodyne 5.0 lL injection loop mounted in a Unijet model

CC-6 cabinet (Bioanalytical Systems, West Lafayette, IN, USA).

The mobile phase was composed of 100 mM sodium acetate,

0.5 mM EDTA, 5 mM sodium octanesulfonic acid, 10 mM NaCl

and 6% acetonitrile; pH 4.0 adjusted with glacial acetic acid. The

mobile phase was briefly bubbled with helium to de-oxygenate it.

The column was maintained at room temperature and the flow

rate was 75 lL/min. DA was detected with a BAS Unijet radial-

flow detector cell with a 6-mm glassy carbon electrode

(Bioanalytical Systems, West Lafayette, IN, USA) coupled to a

model 400 amperometric detector (EG & G Princeton Applied

Research, Princeton, NJ, USA) via an external cell cable. The

applied potential was set at + 450 mV with a sensitivity setting

of 0.5 nA/V. The use of the Unijet reference electrode required an

applied potential setting that was 100 mV less than the equivalent

potential setting for a standard Ag/AgCl reference electrode. The

output of the detector was sent to a Chrom Perfect (Justice

Innovations, Palo Alto, CA, USA) chromatography data analysis

system. The lower limit of sensitivity for DA was approximately

0.2 fmol injected onto the column.

Values were not corrected for in vitro probe recovery efficiency,

which was approximately 15% and in close agreement with published

values (Perry and Fuller 1992). To minimize rat to rat variability, data

for individual experiments were normalized and expressed as percent

change from baseline values. Percent baseline levels for each

experiment were calculated as treatment/control · 100. The average

concentration of three stable samples prior to perfusion with one of

the agents (< 10% variation) was considered the control and was

defined as 100%. Data were analyzed using the statistical program

SPSS. As specified in the figure legends, data were analyzed by two-

way ANOVA, followed by post hoc Tukey’s honestly significant

difference (HSD) test for multiple comparisons unless otherwise

stated. The significance level was set at p < 0.05. The details of the

statistical analysis are contained in the figure legends.

648 S. Rahman and W. J. McBride


Results

Only data from animals that had both probes correctly

implanted in the ACB and VTA were included in this study.

Most (approximately 80%) of the animals that had under-

gone surgery had probes correctly implanted in both sites.

Figure 1 shows representative placements in the ACB and

VTA; overlapping probe placements are not shown. There-

fore, this figure is not a complete quantitative representation

of the distribution of probe placements. Within the ACB,

almost all of the probes perfused both the core and shell to

varying degrees with some placements mostly in the shell,

and some placements mostly in the core. A few probes had

tips close to the olfactory tubercle. Because such a small

portion of the active membrane is exposed to tissue outside

the ACB, it is likely that DA collected in the dialysis

samples is mainly from the core and shell combined. Within

the VTA, a significant portion of the active membrane was

located dorsal to the VTA, and no probes were located in

the substantia nigra. Our previous unpublished data indicate

that extracellular levels of DA were not detected unless a

significant portion of the probe was located within the

VTA, suggesting that the DA detected in the microdialysis

samples originated from the VTA.

Local application of ACSF (n ¼ 5) through the micro-

dialysis probe in the ACB over the same course as the DA

uptake inhibitor or antagonists did not alter the extracellular

levels of DA in the ACB (101 ± 2% of baseline) or the VTA

(105 ± 8% of baseline). These data are consistent with

previous studies (Campbell et al. 1996; Kohl et al. 1998).

Local perfusion of 100 lM GBR through the microdialysis

probe in the ACB increased the extracellular levels of DA

in the ACB to a peak of approximately 500% of baseline,

and concomitantly reduced the extracellular levels of DA in

the VTA to 50% of baseline (Fig. 2). The elevation in the

extracellular levels of DA in the ACB and the concomitant

reduction in the VTA were significantly different (post hoc

tests, p < 0.05) than their own respective baselines from

the 20 to the 100-min time point in both regions (Fig. 2).

Local application of 100 lM BIC (Fig. 2) or 100 lM

PHAC (Fig. 3) alone through the microdialysis probe in

the ACB also increased the extracellular levels of DA in the

ACB to 200–400% of baseline, but neither antagonist signi-

ficantly altered the extracellular levels of DA in the VTA

(Figs 2 and 3).

To examine the role of GABA receptors in the ACB

following increased synaptic levels of DA, BIC or PHAC

was coperfused with GBR via the microdialysis probe in the

ACB. Co-perfusion of 100 lM GBR with either 100 lM BIC

or 100 lM PHAC produced an additional increase in the

extracellular levels of DA in the ACB to approximately

800% of baseline (Figs 2 and 3). However, despite the

additional elevation in the synaptic levels of DA in the ACB,

the reduction in the extracellular levels of DA in the VTA

Fig. 1 Representative locations of micro-

dialysis probe placements in the ACB (left

side) and VTA (right side). Overlapping

placements are not shown therefore the

figure does not indicate the complete

quantitative distribution of the placements.

Numbers in the right indicate distance (mm)

from bregma (Paxinos and Watson 1986).

Black lines correspond to the location of the

active membrane area of the microdialysis

probes.



produced by GBR alone was completely prevented by BIC

and partially prevented by PHAC (Figs 2 and 3).

In contrast to the effects of the GABA antagonists on DA

levels in the ACB, local application of 100 lM SCOP or

MEC alone through the microdialysis probe in the ACB did

not significantly alter the extracellular levels of DA in the

ACB (Figs 4 and 5). Neither antagonist alone significantly

altered the extracellular levels of DA in the VTA.

To examine a possible role of cholinergic receptors in the

ACB on the negative feedback pathway, SCOP or MEC

was coperfused with GBR via the microdialysis probe in

the ACB. Co-perfusion of 100 lM GBR with either 100 lM

SCOP or 100 lM MEC reduced the extracellular levels of

DA in the ACB (Figs 4 and 5). In addition, the reduction in

the extracellular levels of DA in the VTA produced by

GBR was prevented by either cholinergic antagonist (Figs 4

and 5).

Discussion

The findings of the present study suggest that (i) within the

ACB, terminal DA release may be under tonic GABA

inhibition mediated by GABAA and possibly GABAB

receptors; (ii) activation of GABAA and possibly GABAB

receptors within the ACB may be involved in regulating the

negative feedback pathway from the ACB to the VTA; and

(iii) within the ACB, terminal DA release is under the tonic

cholinergic excitatory control of muscarinic and nicotinic

receptors. Within the ACB, there are cholinergic and

GABAergic interneurons that interact with the medium spiny

GABAergic output neurons as well as with DA inputs

(reviewed by McGinty 1999; Meredith 1999).

The observation that perfusion with the GABAA receptor

antagonist alone or in combination with the DA uptake

inhibitor increased the extracellular levels of DA in the ACB

Fig. 2 Effects of local perfusion for 60 min with 100 lM GBR 12909

(d), 100 lM GBR plus 100 lM bicuculline (s), and 100 lM BIC alone

(j) on the extracellular levels of DA in the ACB (top panel) and VTA

(bottom panel). All agents were perfused starting at the zero time

point. Data represent the means ± SEM of between five and eight

animals. A two-way ANOVA (treatment · time) with repeated measures

revealed a significant effect of treatment, F2,16 ¼ 9.87, p < 0.002 and

time, F6,96 ¼ 21.6, p < 0.001 for the ACB; and treatment

F(2,16) ¼ 13.5, p < 0.001 and time, F(6,96) ¼ 5.77, p < 0.03 for the

VTA. There was a significant interaction, F12,96 ¼ 6.30, p < 0.01, for

the ACB and, F12,96 ¼ 3.44, p < 0.05, for the VTA. Asterisks indicate

that there were significant differences (p < 0.05) at 60 and 80 min in

the ACB and at 20–100 min in the VTA between GBR alone vs. GBR

plus BIC (Tukey’s HSD test). The basal extracellular levels of DA in the

ACB and VTA were 43 ± 12 and 24 ± 5 fmol/20 min, respectively.


(d), 100 lM GBR plus 100 lM phaclofen (s), and 100 lM PHAC alone

(j) on the extracellular levels of DA in the ACB (top panel) and VTA

(bottom panel). All agents were perfused starting at the zero time

point. Data represent the means ± SEM of between four and six ani-

mals. A two-way ANOVA (treatment · time) with repeated measures

revealed a significant effect of treatment, F2,11 ¼ 5.97, p < 0.05, and

time, F6,66 ¼ 10.5, p < 0.001, for the ACB; and treatment,

F2,11 ¼ 6.55, p < 0.01, and time, F(6,66) ¼ 3.40, p < 0.001, for the

VTA. The interaction for the ACB, F12,66 ¼ 2.24, p ¼ 0.08, was not

significant, whereas the interaction for the VTA just reached signifi-

cant, F12,66 ¼ 2.29, p ¼ 0.05. However, post-hoc tests (Tukey’s HSD)

revealed that there was no significant difference between GBR alone

vs. GBR plus PHAC. The basal extracellular levels of DA in the ACB

and VTA were 24 ± 5 and 19 ± 5 fmol/20 min, respectively.



suggests that the antagonist is blocking local tonic GABA

inhibition of terminal DA release. A less clear-cut effect was

observed for PHAC, the GABAB receptor antagonist. Tonic

inhibition by both types of GABA receptors on terminal DA

release has been reported in the ventral pallidum (Gong et al.

1998). In addition, Yan (1999) reported tonic GABAA

mediated inhibition of terminal DA release in the ACB;

Smolders et al. (1995) reported tonic GABAergic modula-

tion of striatal DA release. Prefrontal cortical DA release also

appears to be regulated by GABAA and GABAB receptors

(Santiago et al. 1993). Within the VTA, DA neurons

projecting to the ACB appear to be regulated by both types

of GABA receptors (Westerink et al. 1996; Ikemoto et al.

1997). Within the ACB, activation of either GABAA or

GABAB receptors inhibits acetylcholine release and ACB

cholinergic interneurons are under tonic GABAA receptor-

mediated inhibition (Rada et al. 1993). In contrast to these

results, one microdialysis study reported that local perfusion

of the GABAA agonist muscimol increased the extracellular

levels of DA and DOPAC in the ACB (Yoshida et al. 1997).

One possible mechanism to explain these apparent conflict-

ing results is that muscimol may be acting at GABAA

receptors on GABA neurons that regulate terminal DA

release via inhibitory GABAB receptors; inhibiting these

GABA neurons would then result in increased terminal DA

release.

With regard to the present results, GABA receptors could

be directly on DA terminals and/or on excitatory inputs

regulating DA release. An indirect tonic GABA inhibition of

terminal DA release could be occurring via cholinergic

interneurons. The data with the cholinergic antagonists

suggest that terminal DA release is under tonic cholinergic

excitatory influence. Cholinergic interneurons within the

ACB and striatum are under GABA-mediated inhibitory


(d), 100 lM GBR plus 100 lM scopolamine (s) and 100 lM SCOP

alone (j) on the extracellular levels of DA in the ACB (top panel) and

VTA (bottom panel). All agents were perfused starting at the zero time

point. Data represent the means ± SEM of five or six animals. A two-

way ANOVA (treatment · time) with repeated measures revealed a

significant effect of treatment, F2,14 ¼ 15.0, p < 0.001 and time,

F6,84 ¼ 10.7, p < 0.01, for the ACB; and treatment, F(2,14) ¼ 21.9,

p < 0.001, and time, F6,84 ¼ 4.68, p < 0.05, for the VTA. There was a

significant interaction F12,84 ¼ 4.27, p < 0.05, for the ACB and,

F12,84 ¼ 14.0, p < 0.001, for the VTA. Asterisks indicate that there

were significant differences (p < 0.05) at 20 and 40 min in the ACB

and at 20–120 min in the VTA between GBR alone vs. GBR plus

SCOP (Tukey’s HSD test). The basal extracellular levels of DA in the

ACB and VTA were 22 ± 2 and 17 ± 2 fmol/20 min, respectively.


(d), 100 lM GBR plus 100 lM mecamylamine (s), and 100 lM MEC

alone (j) on the extracellular levels of DA in the ACB (top panel) and

VTA (bottom panel). All agents were perfused starting at the zero time

point. Data represent the means ± SEM of between three and six

animals. A two-way ANOVA (treatment · time) with repeated measures

revealed a significant effect of treatment, F2,10 ¼ 12.1, p < 0.01, and

time, F6,60 ¼ 12.6, p < 0.001, for the ACB; and treatment,

F2,10 ¼ 4.75, p < 0.05, and time, F6,60 ¼ 3.47, p < 0.05, for the VTA.

There was a significant interaction for the ACB, F12,60 ¼ 6.34,

p < 0.001, and VTA, F12,60 ¼ 2.25, p < 0.05. Asterisks indicate that

there were significant differences (p < 0.05) at 20 min in the ACB and

at 20 and 40 min in the VTA between GBR alone vs. GBR plus MEC

(Tukey’s HSD test). The basal extracellular levels of DA in the ACB

and VTA were 24 ± 5 and 17 ± 1 fmol/20 min, respectively.



control, involving both GABAA and GABAB receptors,

although tonic GABA inhibition appears mainly under the

influence of GABAA receptors (Anderson et al. 1993; Rada

et al. 1993; DeBoer and Westerink 1994). Therefore, block-

ing tonic GABA inhibition with a GABAA receptor antag-

onist will increase the activity of the cholinergic

interneurons. The present findings suggest that terminal DA

release is under tonic excitatory control by both muscarinic

and nicotinic receptors, because coperfusion with cholinergic

antagonists for either subtype of receptor reduced the GBR-

induced elevation in the synaptic levels of DA. In agreement

with these findings, local perfusion with muscarinic or

nicotinic receptor agonists has been shown to increase

terminal DA release (Marshall et al. 1997; Smolders et al.

1997; Wonnacott et al. 2000). The GABA regulation of the

cholinergic interneurons could come from collaterals of the

medium spiny GABA output neurons or via GABA inter-

neurons (Meredith 1999), or from GABA neurons projecting

from the ventral pallidum (Kalivas et al. 1993b) or VTA

(Van Bockstaele and Pickel 1995).

The observation that a GABAA receptor antagonist (and to

a lesser extent the GABAB antagonist) prevented the

reduction in somatodendritic release of DA in the VTA,

despite further increasing the synaptic levels of DA in the

ACB, suggests that activation of GABAA and possibly

GABAB receptors are required for the long-loop negative

feedback system to operate. Previous studies (Rahman and

McBride 2000, 2001) demonstrated that both D1- and D2-

like receptors were required to activate the long-loop

negative feedback system, as evidenced by reduced somato-

dendritic DA release in the VTA. However, the large

majority of neurons projecting from the ACB to the VTA

express mRNA for the D1 receptor with only a small

percentage expressing mRNA for the D2 receptor (Lu et al.

1998). Expression of D2 receptor mRNA has been found in

neurons projecting from the ACB to the ventral pallidum

(Lu et al. 1998), as well as in cholinergic interneurons

(MacLennan et al. 1994; Jongen-Relo et al. 1995). There-

fore, if a co-operative interaction of D1 and D2 receptors is

required to activate the feedback pathway from the ACB to

the VTA, this interaction could involve other neuronal

elements then just the medium spiny GABA neurons

projecting to the VTA. The present data suggest that

cholinergic interneurons, and GABA interneurons or GABA

collaterals from medium spiny neurons projecting to the

ventral pallidum may also have a role in regulating the long-

loop feedback pathway.

Cholinergic interneurons may be involved in the ACB

mechanisms underlying feedback control because addition of

either a muscarinic or nicotinic receptor antagonist reduced

terminal DA release and prevented the reduction in somato-

dendritic DA release in the VTA produced by GBR alone

(Figs 4 and 5). However, the influence of the cholinergic

interneurons may be at the level of regulating terminal DA

release. Reducing the synaptic levels of DA in the ACB with

SCOP and MEC may have been sufficient to prevent

activation of the feedback pathway. A previous study

indicated that increasing synaptic DA levels to 150–200%

of baseline did not affect the feedback pathway as evidenced

by no change in the somatodendritic release of DA in the

VTA (Rahman and McBride 2000). On the other hand,

increasing the extracellular DA levels in the ACB to 400% or

higher of baseline did activate the feedback pathway

(Rahman and McBride 2000). In addition, the cholinergic

receptor antagonists could also be acting at receptors on

GABA output neurons to prevent activation of the feedback

pathway (Di Chiara et al. 1994; Harsing and Zigmond 1998).

The finding that BIC completely blocked the effects of

GBR in reducing somatodendritic DA release in the VTA

suggests that there may be a GABA–GABA neuronal

interaction within the ACB in the feedback pathway. D1–

D2 interactions could result in the activation of the first

GABA neuron and the subsequent inhibition of the second

GABA neuron, which projects to the VTA. Inhibition of the

ACB medium spiny GABA output neuron releases the VTA

GABA inhibitory interneuron from tonic inhibition. Disin-

hibiting the VTA GABA inhibitory interneuron enhances its

inhibitory influence on the VTA DA neuronal activity

resulting in reduced somatodendritic DA release. There is

evidence of tonic GABAA receptor mediated inhibition of

VTA DA neurons projecting to the ACB (Westerink et al.

1996; Ikemoto et al. 1997). Local perfusion of the ACB with

BIC blocks the negative feedback loop suggesting that

GABAA receptors on GABA output neurons are involved in

the feedback pathway. This may also be the case for GABAB

receptors, although it may occur to a lesser degree. The

activation of the first GABA neuron (interneuron or collateral

from medium spiny neurons) may involve excitatory D1 and

inhibitory M4 receptors on these neurons (see Di Chiara

et al. 1994). The cholinergic input on these neurons at the

M4 receptor could be controlled by inhibitory D2 receptors

on these terminals (Di Chiara et al. 1994). Concurrent

activation of D1 and D2 receptors is required to activate this

first GABA neuron. Activation of D2 receptors decreases

acetylcholine release and reduces inhibition at M4 receptors

while concurrent activation of D1 receptors results in

increased GABA release. The increased GABA release

inhibits the GABA output neuron projecting to the VTA

resulting in disinhibition of VTA GABA inhibitory inter-

neurons.

The 100 lM concentration of each antagonist was chosen

because the results of previous microdialysis studies indi-

cated that this concentration produced reliable effects which

were reasonably selective (see statements in Methods

section). However, because significantly lower concentra-

tions of the antagonists were not tested and the concentration

of the antagonist in the extracellular space is unknown, it is

possible that the antagonists may also be acting non-



selectively at other receptors, which could contribute to the

observed effects.

In summary, the present findings suggest that GABA–

GABA neuronal interactions within the ACB are involved in

the feedback system regulation VTA DA neuronal activity. In

addition, the data suggest that cholinergic mediation of

terminal DA release within the ACB may play a role in

regulating the negative feedback pathway to the VTA.

Acknowledgement

This work was supported in part by PHS grant AA10721.

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