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Keywords:

  • cholinergic receptors;
  • dopamine;
  • feedback regulation;
  • GABA receptors;
  • nucleus accumbens;
  • ventral tegmental area

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

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 tegmental area (VTA). Adult male Wistar rats were implanted with ipsilateral dual guide cannulae for in vivo microdialysis studies. Activation of the feedback system was accomplished by perfusion of the ACB with the DA uptake inhibitor GBR 12909 (GBR; 100 µm). To assess the involvement of GABA and cholinergic receptors in regulating this feedback system, antagonists (100 µm) 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 interneurons may influence the negative feedback system by regulating terminal DA release within the ACB.

Abbreviations
used

ACB, nucleus accumbens

ACSF

artificial cerebrospinal fluid

BIC

bicuculline

DA

dopamine

MEC

mecamylamine

PHAC

phaclofen

SCOP

scopolamine

VTA

ventral tegmental area.

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 via a long-loop negative feedback system. These microdialysis 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 interneurons) 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 somatodendritic 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

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

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 institutional 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 rostral-caudal direction and extended approximately 500 µm. The outside diameter of the dialysis membrane was 220 µm. 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 microdialysis, 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. Experiments 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 µL/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-µm 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 µL 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 µL 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 µm concentration used for the antagonists 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 µm 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 µm coronal 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 electrochemical detection system as described (Rahman and McBride 2000) to determine DA levels in each sample. Briefly, chromatography was performed using a model 2350 pump (ISCO, Lincoln, NE, USA) with a BAS SepStik microbore analytical column (1.0 × 100 mm column; 3 µm Spherisorb C18 stationary phase) connected to a BAS custom injection valve and a Rheodyne 5.0 µL 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 µL/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.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

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 undergone surgery had probes correctly implanted in both sites. Figure 1 shows representative placements in the ACB and VTA; overlapping probe placements are not shown. Therefore, 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.

image

Figure 1. Representative locations of microdialysis 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.

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Local application of ACSF (n = 5) through the microdialysis 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 µm 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 µm BIC (Fig. 2) or 100 µm 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␣significantly altered the extracellular levels of DA in the VTA (Figs 2 and 3).

image

Figure 2. Effects of local perfusion for 60 min with 100 µm GBR 12909 (●), 100 µm GBR plus 100 µm bicuculline (○), and 100 µm BIC alone (▮) 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 = 9.87, p < 0.002 and time, F6,96 = 21.6, p < 0.001 for the ACB; and treatment F(2,16) = 13.5, p < 0.001 and time, F(6,96) = 5.77, p < 0.03 for the VTA. There was a significant interaction, F12,96 = 6.30, p < 0.01, for the ACB and, F12,96 = 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.

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image

Figure 3. Effects of local perfusion for 60 min with 100 µm GBR 12909 (●), 100 µm GBR plus 100 µm phaclofen (○), and 100 µm PHAC alone (▮) 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 animals. A two-way anova (treatment × time) with repeated measures revealed a significant effect of treatment, F2,11 = 5.97, p < 0.05, and time, F6,66 = 10.5, p < 0.001, for the ACB; and treatment, F2,11 = 6.55, p < 0.01, and time, F(6,66) = 3.40, p < 0.001, for the VTA. The interaction for the ACB, F12,66 = 2.24, p = 0.08, was not significant, whereas the interaction for the VTA just reached significant, F12,66 = 2.29, p = 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.

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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 µm GBR with either 100 µm BIC or 100 µm 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 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 µm 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.

image

Figure 4. Effects of local perfusion for 60 min with 100 µm GBR 12909 (●), 100 µm GBR plus 100 µm scopolamine (○) and 100 µm SCOP alone (▪) 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 = 15.0, p < 0.001 and time, F6,84 = 10.7, p < 0.01, for the ACB; and treatment, F(2,14) = 21.9, p < 0.001, and time, F6,84 = 4.68, p < 0.05, for the VTA. There was a significant interaction F12,84 = 4.27, p < 0.05, for the ACB and, F12,84 = 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.

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image

Figure 5. Effects of local perfusion for 60 min with 100 µm GBR 12909 (●), 100 µm GBR plus 100 µm mecamylamine (○), and 100 µm MEC alone (▪) 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 = 12.1, p < 0.01, and time, F6,60 = 12.6, p < 0.001, for the ACB; and treatment, F2,10 = 4.75, p < 0.05, and time, F6,60 = 3.47, p < 0.05, for the VTA. There was a significant interaction for the ACB, F12,60 = 6.34, p < 0.001, and VTA, F12,60 = 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.

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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 µm GBR with either 100 µm SCOP or 100 µm 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

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

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 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 modulation 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 conflicting 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 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, blocking tonic GABA inhibition with a GABAA receptor antagonist 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 interneurons (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 somatodendritic 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). Therefore, 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 somatodendritic 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. Disinhibiting 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 interneurons.

The 100 µm concentration of each antagonist was chosen because the results of previous microdialysis studies indicated that this concentration produced reliable effects which were reasonably selective (see statements in Methods section). However, because significantly lower concentrations 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

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

This work was supported in part by PHS grant AA10721.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References
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