Dopaminergic involvement in medial prefrontal cortex and core of the nucleus accumbens in the regulation of ethanol self-administration: a dual-site microinjection study in the rat

Physiology & Behavior 79 (2003) 581 – 590

Dopaminergic involvement in medial prefrontal cortex and core of the nucleus accumbens in the regulation of ethanol self-administration: a dual-site microinjection study in the rat Herman H. Samson*, Ann Chappell Center for the Neurobehavioral Study of Alcohol, Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, NC 27157-1083, USA Received 18 October 2002; received in revised form 2 April 2003; accepted 7 April 2003

Abstract The complex mesolimbic – mesocortical system involved with behavioral selection has been implicated in the control of ethanol selfadministration. However, the nature of the interactions within this multiple-structured system in ethanol intake regulation remains unclear. Although the role of dopamine (DA) in the prefrontal cortex and the nucleus accumbens has been examined individually, the interaction of DA activity in both structures at the same time remains to be examined. Male, Long – Evans rats were initiated to self-administer ethanol in an operant situation using the sucrose-substitution procedure. Following initiation, bilateral cannula guides were located to allow microinjection in the medial prefrontal cortex (mPFC) and the core of the nucleus accumbens. The DA D2/D3 agonist quinpirole (10.0-mg dose in the prefrontal cortex; 4.0-mg dose in n. accumbens) and the D2 antagonist raclopride (0.05-mg dose in prefrontal cortex; 1.0-mg dose in the nucleus accumbens) were then tested in each site alone and in combination in both sites in each rat. Changes in total responding, ethanol intake, and the pattern of responding were analyzed. Single-site injections replicated most of our previous findings for these doses. Changes in single-site effects were found when dual-site injections were performed, with altered input from the prefrontal areas impacting the effects of accumbens injections. Based on these interactions, our hypothesis that the prefrontal area is involved with the onset and offset of drinking, while the nucleus accumbens is involved with maintaining the ongoing behavior, remains viable. D 2003 Elsevier Inc. All rights reserved. Keywords: Medial prefrontal cortex; Core of the nucleus accumbens; Dual microinjection; Ethanol; Rat

1. Introduction The involvement of the rodent’s mesolimbic –mesocortical system in ethanol self-administration has been clearly demonstrated over the last 15 years [1,2]. Almost all the brain areas that are interconnected in this system [3– 5] have been shown to be involved to some extent in ethanol selfadministration behavior. While the dopaminergic (DA) projection from the ventral tegmental area (VTA) to the nucleus accumbens core (nAcc core) has been the most studied (see Ref. [2] for a review), other anatomical structures in this system, including the medial prefrontal cortex (mPFC) [6,7], the amygdala [8], and the pedunculopontine

* Corresponding author. Tel.: +1-336-716-8590; fax: +1-336-7137168. E-mail address: [email protected] (H.H. Samson). 0031-9384/03/$ – see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0031-9384(03)00126-4

nucleus (PPN) [9,10], have been implicated in the control of ethanol self-administration behavior. One neurotransmitter in this system that has received major attention is the role of DA transmission in the VTA, nAcc core, and mPFC [6,11– 18]. Both the nAcc core and mPFC receive DA enervation from the VTA [3,19,20]. The mPFC has a glutaminergic projection that innervates the nAcc and the VTA [20]. The nAcc core has projections to the ventral pallidum, which then innervates the medial thalamus that has connections back to the mPFC [3,21], in addition to projections to the VTA. Thus, a complex network of feed-forward and feedback loops within this part of the mesolimbic system exists between the mPFC, nAcc core, and VTA. It has been hypothesized that DA transmission plays an important role in the integration of information processing in this part of the mesolimbic system [3,19,20,22] and, therefore, in the control of many types of self-administration behavior [23].

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Dopamine transmission in the mPFC appears to modulate the activity of the pyramidal neurons (for a review, see Ref. [20]). This modulation results from a direct action on the pyramidal cell body and from an action on the inhibitory GABA interneurons that also regulate the activity of the pyramidal cells. Thus, DA activity is part of a complex neurotransmission integration system within the mPFC. Dopamine in the nAcc core modulates the activity of the GABAergic spiny output neurons [24,25]. This is the result of an inhibitory action on the spiny cells, which is integrated along with the excitatory input received in part from the mPFC, hippocampus, and hypothalamus. Therefore, there is a complex interaction between the VTA DA input to both the mPFC and nAcc core and the mPFC input to the nAcc. This forms a loop system believed to integrate the activity within this portion of the mesolimbic circuitry [3,19]. While there are data on the effects of manipulation of DA transmission on ethanol self-administration within either the nAcc core or the mPFC, there has been no attempt to comanipulate DA transmission concurrently in these structures. In order to examine how the complex interaction of DA activity in these brain areas might be involved in ethanol self-administration, the following study was designed using concurrent microinjection of DA agonists or antagonists into the mPFC and the core of the nAcc before the onset of an operant ethanol self-administration session.

2. Methods 2.1. Animals Twelve adult, Long –Evans male rats (Harlan, Indianapolis) were used for these studies. Upon arrival in the laboratory, they weighed 231 ± 3 g and were individually housed in hanging stainless steel cages with food and water available ad lib. Artificial lighting was on for 12 h/day, starting at 0600 h. The animals were kept in an AAALAC approved animal facility, with temperature maintained in accordance with NIH Animal Use and Care Guidelines. All experimental procedures were approved by the Wake Forest University School of Medicine Animal Care and Use Committee. 2.2. Apparatus All operant sessions were performed in Med Associates (St. Albans, VT) operant chambers equipped with one lever and one dipper fluid-delivery system. Experimental control and data acquisition was with a personal computer interfaced with Med Associates control units and programmed using MED-PC (Med Associates). Microinjections were performed using Harvard microsyringe pumps (Model 22, Natick, MA) and Hamilton 1.0 ml syringes (Reno, NV).

2.3. Procedure Upon arrival in the laboratory, the rats were given 2 days to adapt to the housing conditions. They were handled and weighed each day. For the next 3 days, the only available fluid on the home cage was 10% ethanol (10E), provided in a calibrated drinking tube attached to the front of the cage. Intakes and body weights were recorded daily. Following these 3 days of forced ethanol drinking, the animals were given 14 days of a two-bottle fluid-choice procedure, with 10E available in one drinking tube and water in the other tube. Intakes and body weights were recorded daily and an ethanol preference ratio calculated (milliliters of 10E/total fluid intake in milliliters). The tubes were alternated randomly on the front of the cage to decrease the effect of tube or side preference. The preference ratio, along with ethanol intake in grams per kilogram, was used as a measure of initial ethanol consummatory behavior, before any operant training. This home cage testing has been a standard feature of our ethanol-initiation procedure for many years. It provides initial information about each cohort of rats in relation to prior studies. It may also be important for the initiation procedure in terms of acquainting the rats with the taste of ethanol. Following the home cage preference test, the rats were trained to self-administer ethanol in the operant chambers using the sucrose-substitution procedure [26]. The rats were water deprived overnight and then placed into the operant chambers where they were shaped to lever press using 20% sucrose (20S) as the reinforcing solution presented in the dipper. When lever pressing was shaped, the rats were then given an overnight session in the operant chambers with the 20S presented in the dipper for 3 s following each lever press. After this single overnight session, the rats were returned to their home cages with food and water available. From this time on, no food or water restriction was employed. On the following day, the rats were given a 30min session in the operant chamber with 10S presented in the dipper following each lever press. From this time on, the rats received one daily 30-min session in the operant chambers, 5 days/week. Over the next 14 sessions, ethanol was added in increasing concentrations and sucrose decreased in concentration until on the 14th session, 10E in water was the fluid presented in the dipper. During this time, the schedule of reinforcement remained at a fixed ratio of one (FR 1). Over the next 12 sessions, the schedule of reinforcement was incremented to 2, then 3 and then to 4 by the 12th session. This FR4 schedule was in effect for the duration of the experiment. The rats then received 26 sessions to stabilize their ethanol self-administration before surgery. The mean of the last 5 of these 26 sessions were used as a presurgery baseline measure of responding and intake. On the day of surgery, no session was given. Daily sessions were resumed the day after surgery, with the FR

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requirement reduced to 1. On the following day and for the rest of the study, the FR was set at four responses. The next 18 sessions were identical to the baseline sessions before surgery. The mean of the last five of these sessions were used as the postsurgery baseline and were compared to the presurgery baseline to determine any effect that surgery may have had on self-administration behavior. Only rats that recovered to within 15% of their presurgery baseline were continued in the study. The following experimental protocol was then used to determine the effects of the microinjections. The Monday and Tuesday sessions were identical to those used in baseline, with the Tuesday session designated as the no injection (NI) session for that series of five sessions. On Wednesday, the rats received a sham injection 10 min before their session, and on Thursday the rats received a microinjection 10 min before the session. The Friday session was the same as Monday and Tuesday and was used to determine if any carryover effects from the Thursday injection occurred. The following microinjection schedule was employed, with one injection tested during each weekly session series. The first microinjection was 1.0 mg raclopride into the nAcc core. Next, the effect of 10 mg quinpirole in the mPFC was examined. This was followed by the dual microinjection of 1.0 mg raclopride in the nAcc and 10 mg quinpirole in the mPFC. The next microinjection was 4 mg quinpirole in the nAcc. This was followed by the dual microinjection of 4 mg quinpirole in the nAcc and 10 mg quinpirole in the mPFC. The next microinjection was the dual injection of 0.05 mg raclopride in the mPFC and 4 mg quinpirole in the nAcc. Next, the microinjection of 0.05 mg raclopride in the mPFC was tested. Last, the dual microinjection was 0.05 mg raclopride in the mPFC and 1.0 mg raclopride in the nAcc. Following the last session, the animals were sacrificed and their brains removed for the determination of the microinjection sites. 2.4. Surgery The rats were anesthetized with sodium pentobarbital (40 mg/kg ip) and placed into a Kopf stereotaxic instrument (Tujunga, CA). The nose bar was set at 3.3 mm. Coordinates for placement of the guide cannula to end 1.0 mm above the injection point in the mPFC were AP: + 2.7 mm; ML: 1.95 mm; DV: 2.5 mm at a 16 angle to the midline and for the nAcc core were AP: + 1.5 mm; ML: 1.8 mm; DV: 6.0 mm. These coordinates were taken from the Paxinos and Watson rat brain atlas [27]. After a midline incision was made over the skull, the skull was scraped clean and allowed to air-dry for approximately 5 min. Small holes were drilled into the skull and selftapping stainless steel screws placed to secure the cranioplastic cement (Plastics One, Roanoke, VA) to the skull. Next, the sites for the cannula guides were marked and holes drilled through the skull for the bilateral placement of

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the 26-gauge stainless steel guides. Once in place, the guides were attached to the skull using the cranioplastic cement. When the cement was dry, the animal was removed from the stereotaxic instrument, and sterile injectors (33 gauge), 1 mm longer than the guides, were inserted into the brain and removed after 1 min. This was done to make the initial brain penetration while the animal was still anesthetized. Then sterile obturators, the same length as the guide cannula, were inserted into the guides to prevent them from clogging and to reduce the potential for brain infection. The rats were placed into a plastic cage under a heat lamp and kept under supervision until they had recovered from the anesthesia. The obturators were checked daily, cleaned with a sterile saline solution and replaced as needed. 2.5. Microinjection procedure Bilateral, simultaneous injections were given to the rats 10 min (including the injection time) before the start of a session. The rats were gently hand-held while the obturators were removed and the injectors were inserted. The injectors were 1 mm longer than the guide cannula. Once the injectors were inserted, the rats were placed into a small plastic tub and allowed to move freely during the infusion. The infusion pumps were operated for 1 min, delivering 0.5 ml of drug/injector (a total of 1.0 ml into the brain). The injectors were left in the brain for an additional minute after the end of the injection before being removed and the sterile obturators replaced. Following the injection, the rats were placed into a transport cage, where they remained until the start of the session. As noted above, only one injection was given per week. For single-site injections, only one set of injectors was used, while for dual-site injections, four injectors were placed at the same time and both sites injected at the same time. Sham injections were performed weekly as a control for the injection procedure. This procedure was the same as a drug injection, except that the injectors used were the same length as the cannula guides and the syringes were not placed in the infusion pumps. For dual-site injections, dual sham procedures were used. These sham injections were used as the control condition for the microinjections, as they do not result in any additional brain injury and have been shown to be identical to vehicle control injections [17]. Given the limited number of injections available before potentially compromising brain damage (we have found that between 8 and 10 injections result in only moderate glial formation at the injection site [18]), no vehicle injections were tested. This allowed for the single and combination microinjections of each drug at each site to be tested without exceeding the number of injections that could compromise the results. This omission of the vehicle control was based on our prior work that had shown no effect of the microinjection of the artificial cerebrospinal fluid vehicle [17].

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2.6. Drugs

3. Results

Sucrose solutions were prepared weight/volume, and ethanol solutions were prepared as volume/volume. Raclopride (Astra, Sweden) and Quinpirole (RBI, Natick, MA) were dissolved in artificial cerebrospinal fluid and shaken on a mechanical shaker to assure complete incorporation into solution. The solutions were then sterile filtered (Millipore 0.2-mm filters, Gellman). All microinjection solutions were prepared immediately before injection. The doses of raclopride and quinpirole were chosen based on our previous work with these drugs in these areas that had been to alter ethanol self-administration [6,14].

3.1. Home cage ethanol intake and preference ratio

2.7. Histology At the end of the experiments, the rats were given a lethal dose of sodium pentobarbital (80 mg/kg ip) and when deeply anesthetized were transcardially perfused with buffered saline, followed by 10% formalin in saline. The brains were removed and stored in 10% formalin for at least 1 week before being sectioned on a freezing microtome (60-mm sections), mounted on slides, and stained with cresyl violet. Location of the injection sites was determined using a light microscope. 2.8. Data analysis Daily session intakes in milliliters were determined on the basis of the number of dipper presentations and the volume of fluid change in the dipper reservoir. Body weights were used to calculate ethanol intake in grams ethanol per kilogram of body weight (g/kg). Total responses and reinforcements for each session were recorded for analysis. Cumulative response records were examined visually to evaluate effects of drug administration on the pattern of self-administration. In addition, an analysis program that examined several parameters of the response pattern [28] was employed to provide an objective evaluation of any effects visually observed in the cumulative response records. This microanalysis included the determination of runs of responding that were defined as two or more responses that occurred with less than a 1-min pause between responses. By examining the number, size, and rate of these runs, we have found that changes in the overall pattern of responding can be quantified. The number of responses in the first run (first run size) has been shown to be a descriptive measure of the overall change in response pattern resulting from drug or behavioral manipulations [29]. All values are reported as means and standard error of the means (S.E.M.), unless otherwise noted. A within-subjects repeated-measures analysis of variance (ANOVA) was used to compare no injection, sham injection, and drug data for each drug/site combination, with post hoc testing using the Student – Newman – Keuls procedure. All statistical analyses were done with the use of a commercial statistical package (Sigmastat, SPSS, Chicago, IL).

During the 3 days when only 10E was available as the fluid on the home cage, the rats consumed 6.85 ± 0.12 g/kg/ day (range, 6.3 to 7.6 g/kg). During the following 14 day, two-bottle home cage preference test, the rats consumed 1.47 ± 0.18 g/kg ethanol/day and had a preference ratio (ethanol intake/total fluid intake) of 18.7 ± 2.4%. These values are similar to our previous studies, suggesting this cohort’s initial ethanol acceptability and intake did not differ markedly from the rats used in our prior microinjection studies. As a result of illness, incorrect cannula placement, and unrecoverable behavioral baseline after surgery, 5 of the 12 rats had to be dropped from the study. Thus, the data reported are for the 7 rats that completed all phases of the study. The locations for the injections from these 7 rats are illustrated in Fig. 1. 3.2. Operant self-administration baselines Prior to surgery, following self-administration initiation, the rats made 149 ± 18 responses/session and consumed 0.56 ± 0.07 g/kg ethanol. Following surgery, they made 154 ± 23 responses/session and consumed 0.57 ± 0.08 g/kg. There were no significant differences in either measure between the pre- and postsurgery baseline measures. 3.3. Microinjection results The microinjection of 1.0 mg raclopride in the nAcc core resulted in a significant decrease in responding [ F(6,2) = 56.661, P < .001], an effect previously noted for this dose [14] (Fig. 2). As well, the microinjection of the 10.0-mg dose of quinpirole in the mPFC reduced responding [ F(6,2) = 7.466, P < .008] as previously reported [6] (Fig. 2). When both sites were concurrently microinjected at these doses, there was also a significant reduction in responding [ F(96,2) = 31.642, P < .001]. It appeared that there might be a slight enhancement of the suppression of responding resulting from the dual injection but there was no significantly greater effect when compared to either drug alone. There was no indication that one injection site altered the actions of the injection at the other site. Examination of the cumulative response records indicated that the nature of the response reduction was predominated by an early termination of a normal response pattern. The analysis of response pattern confirmed this, indicating that for all three microinjections trials, the size of the first run of responses was decreased to the same degree in all three conditions, all of which were significantly decreased from their respective shams [paired t tests: raclopride/nAcc core, t(6) = 4.270, P < .005; quinpirole, mPFC, t(6) = 3.161, P < .020; dual injection at both sites/same drug and dose, t(6) = 4.480,

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Fig. 2. Ethanol-maintained responding following microinjections of raclopride in the nAcc core, quinpirole in the prefrontal cortex (mPFC), and concurrent (DUAL) injections at both sites. Doses are in micrograms of the drug. * Significant from the no injection (NI) condition. * * Significant from the sham injection (SHAM) condition. Error bars represent standard error of the means.

in the mPFC (Fig. 3). There were no effects on any response rate measures. However, while total responding was decreased to the same amount by either the quinpirole injection in the mPFC or the dual injection, there was only a significant decrease in first run size following the mPFC injection (Table 1). Thus, the dual injection altered the nature of the effect of the mPFC injection alone on the pattern of responding. The microinjection of 0.05 mg raclopride in the mPFC resulted in a small but significant decrease in responding [ F(6,2) = 6.072, P < .01] (Fig. 4). The dual injection of quinpirole in the nAcc and raclopride in the mPFC resulted in no significant effects upon responding (Fig. 4). Thus, the Table 1 Number of responses in the first run (first run size)

Fig. 1. Anatomical locations of microinjection sites (taken from Paxinos and Watson [27]). nAcc, nucleus accumbens sites. mPFC, prefrontal cortical sites. The numbers represent the distance from bregma based on the rat brain atlas.

P < .004] (Table 1). There was no effect upon either total rate of responding or the rate of responding in the first run for any injection condition. The microinjection of 4 mg of quinpirole in the nAcc had no significant effect upon responding (Fig. 3) (it should be noted that data for single injections are repeated in the figures for purposes of comparison). This did not replicate our previous study that found a small but significant reduction with this dose in this site [14]. The dual injections of quinpirole in the mPFC and nAcc core resulted in a significant reduction in total responding [ F(6,2) = 10.684, P < .002] that was not different from that of quinpirole alone

NI

Sham

nAcc 1.0 mg raclopride 4.0 mg quinpirole

127 (30) 115 (30)

158 (33) 107 (28)

43 (11) * 66 (16)

PFC 0.05 mg raclopride 10.0 mg quinpirole

124 (35) 177 (33)

103 (20) 112 (24)

83 (27) 32 (14) *

Dual nAcc nAcc nAcc nAcc

86 (15) 104 (24) 114 (28) 123 (29)

rac/PFC rac rac/PFC quin quin/PFC quin quin/PFC rac

83 96 79 98

(14) (19) (19) (27)

Drug

45 16 55 127

(18) (6) * (25) (68)

Values are means with S.E.M. in parenthesis. Doses for the dual injections are the same as used for each site alone. NI, no injection control; nAcc, nucleus accumbens; PFC, prefrontal cortex; rac, raclopride at the dose for that site; quin, quinpirole at the dose for that site. * The drug value is significantly different from sham at P < .05 or greater.

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Fig. 3. Ethanol-maintained responding following microinjections of quinpirole in the nAcc core, quinpirole in the prefrontal cortex (mPFC), and concurrent (DUAL) injections at both sites. Doses are in micrograms of the drug. * Significant from the no injection (NI) condition. * * Significant from the sham injection (SHAM) condition. Error bars represent standard error of the means.

nAcc quinpirole injection antagonized to some degree the effects of raclopride in the mPFC. However, the variability and small effect of raclopride injected alone in the mPFC makes the interpretation of these data difficult. No rate effects or effects on first run size were observed for any of these injections. The dual microinjection of 1.0 mg raclopride in the nAcc and 0.05 mg raclopride in the mPFC resulted in a significant reduction of total responding [ F(6,2) = 5.059, P < .02] (Fig. 5). Using percent of sham responding for the three conditions, a one-way repeated measures analysis of variance found a significant difference between the conditions (raclopride/nAcc, 27 ± 5% of sham, raclopride/mPFC,

Fig. 5. Ethanol-maintained responding following microinjections of raclopride in the nAcc core, raclopride in the prefrontal cortex (mPFC), and concurrent (DUAL) injections at both sites. Doses are in micrograms of the drug. * Significant from the no injection (NI) condition. * * Significant from the sham injection (SHAM) condition. Error bars represent standard error of the means.

87 ± 10% of sham, Dual injection, 66 ± 13% of sham) [ F(6,2) = 20.458, P < .001]. A pairwise comparison found that the injection of raclopride in the mPFC alone and in combination with raclopride in the nAcc were not different from each other but both were different from raclopride injected in the nAcc alone, suggesting a partial reversal of the nAcc effect by the dual mPFC injection. There were no effects of any of the injections upon rate of responding. When the size of the first run was compared for the three conditions, suppression of first run size by raclopride following the nAcc core injection was not different from that resulting after the dual injection, while both were significantly decreased compared to raclopride injected in the mPFC alone [ F(2,12) = 4.332, P < .038; post hoc test significant at 0.05].

4. Discussion

Fig. 4. Ethanol-maintained responding following microinjections of quinpirole in the nAcc core, raclopride in the prefrontal cortex (mPFC), and concurrent (DUAL) injections at both sites. Doses are in micrograms of the drug. * Significant from the no injection (NI) condition. * * Significant from the sham injection (SHAM) condition. Error bars represent standard error of the means.

The effects following single-site microinjections replicated our prior findings, with the exception of the microinjection of quinpirole in the nAcc core. Previously, we had observed a small but significant reduction in ethanol-maintained responding compared to sham injections with the 4.0mg dose of quinpirole in the nAcc core [14]. Although not statistically significant, a similar small decrease in the response pattern was observed in the present study. Because the effects of the single doses tested generally replicated our prior work, they appear to have been appropriate for this initial dual-injection study. Clearly, different dose combinations could provide different results. However, given the limitations of the number of microinjections that can be performed at a given site, we feel that the data of this study provided an important beginning in examining the interac-

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tions between these brain areas relating DA activity to ethanol self-administration. The application of the DA D2 antagonist raclopride in the nAcc core should have affected most of the D2 receptors occurring in this structure, and the concurrent injection of the DA D2/D3 agonist quinpirole in the mPFC should have been sufficient to impact a large portion of D2/ D3 receptors at that site. Separately, injections of these agents at either site significantly reduced responding maintained by ethanol presentation (Fig. 2). Analysis of the response patterns suggested that the nature of the reduction of responding was similar for both injections, with no effect on momentary response rate but an early termination of responding compared to sham conditions. This reduction in responding was primarily a result of reducing the size of the first run of responding. This pattern of change in responding suggests that blocking of the DA D2 activity in the nAcc core or facilitating DA D2/D3 activity in the mPFC has very similar effects on responding maintained by ethanol presentation. These effects did not appear to facilitate each other when a dual injection was performed at the doses tested (Fig. 2). It is possible that at lower doses of either agent at either site, facilitation might be observed. We have previously speculated that one possible action of the microinjection of quinpirole into the mPFC would be to result in a decrease in DA function in the nAcc core [6]. If this occurred, then the net behavioral result of either a DA antagonist in nAcc or the DA agonist in the mPFC would be similar in nature, in that in either case DA transmission in the nAcc would be decreased. The lack of a greater effect of the dual injection would suggest that the extent of the decrease in DA transmission following either site injection had reached maximum at the doses tested. However, the glutamergic input from the mPFC not only impacts DA release in the nAcc [30,31] but also has direct effects on the activation of the nAcc core GABAergic spiny cells. Thus, the effect of quinpirole in the mPFC on responding could be involved with decreased stimulatory activity on the nAcc spiny neurons in addition to changes in DA transmission. The data support a hypothesis that changes in information feedback via either a direct shift of input from the mPFC or from DA activity in the nAcc arising from the VTA can result in an early termination of responding maintained by the presentation of ethanol. Most likely, during ‘‘normal’’ behavioral situations, it is a combination of all these actions that regulate the duration of a given drinking bout. Because there was no significant effect of the 4.0-mg dose of quinpirole injected in the nAcc core, the effect of the dual quinpirole injections in the mPFC and the nAcc appeared to be the same as the mPFC effect alone. However, analysis of the first run size found that whereas the quinpirole injection in the mPFC decreased first run size and the quinpirole injection in the nAcc had no effect, the dual injection of quinpirole in both sites had no effect on first run size (Table 1). Thus, while the extent of the

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decrease in total responding was the same for quinpirole alone in the mPFC and following the dual quinpirole injections, the pattern of the decrease in responding was different between the two conditions, indicating that there was an interaction following the dual injection. It would appear that the activation of DA transmission in the nAcc core by this dose of the DA agonist quinpirole, although not resulting in a significant change in responding by itself, can partially counter the effect of the mPFC injection. This suggests that an important effect of the mPFC quinpirole injection in reducing ethanol-maintained behavior could be related to a decrease of DA release in nAcc [31]. By enhancing DA activity by the concurrent quinpirole injection in the nAcc core, the maintenance of the initial portion (first run) of the self-administration pattern could result. However, this DA agonist effect in the nAcc core was not sufficient to block the decrease of total responding when compared to sham conditions. If the initial portion of the response pattern of self-administration is related to a feedforward control process involving DA release in the nAcc core, perhaps related to the extent of mPFC input activity, while overall termination of a self-administration bout (as assessed by total session responding) is also a function of glutamatergic input from the mPFC, then the effects observed following this combination of dual microinjections would occur. We have suggested that the onset and offset of self-administration behavior is under partial control of mPFC input to the nAcc core (along with the inputs from other areas to the mPFC, i.e., the amygdala, hippocampus, and hypothalamus), while the maintenance of self-administration once started is a function of VTA feedback activity to both structures [2]. The finding of the effect of the dual injection of quinpirole in the mPFC and nAcc core on the pattern of responding appears to support this hypothesis. Although the microinjection of DA D2 antagonist raclopride in the mPFC also decreased total responding like the injection of the DA agonist, the effect was much smaller and the result of a different change in the response pattern [14]. In the Hodge et al. study [14], the effect of the agonist was to decrease the time spent responding (early termination) with no effect on the rate of responding. When raclopride was injected in the mPFC, Hodge et al. [14] observed a reduction in the rate of responding, with only a small effect on total responses. However, in the present study, there was no significant effect of raclopride in the mPFC on either rate or extent of responding when compared to sham injections. Although it appeared that the concurrent injection of quinpirole in the nAcc blocked the very small effect of the raclopride in the mPFC (Fig. 4), the variability across animals resulted in no statistically significant results. Thus, it is impossible to determine exactly what the role of DA antagonism within the mPFC might have in combination with the effects of a DA agonist injected in the nAcc core. Further studies using different doses will be required to better assess this interaction.

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However, whereas raclopride injected alone into the mPFC had no effect compared to sham injections, it resulted in a significant block of the response reduction that resulted from the raclopride injection into the nAcc core (Fig. 5). Thus, the effect of the raclopride injection into the mPFC, when combined with the concurrent injection of raclopride in the nAcc demonstrated the importance of DA activity in the mPFC on behavioral regulation within this mesolimbic – mesocortical system—an effect that would have been undetected had only single-site injection procedures been used. The nature of the changes in the pattern of responding between the single injections and the dual injection was similar to that observed when quinpirole was injected into each area. However, for raclopride, the ability of the mPFC injection to impact the size of the nAcc raclopride effect on first run size was much greater than that observed when quinpirole was the injected drug. These data are additional support for the hypothesis that the input from the mPFC to the nAcc provides control over the onset and offset of a selfadministration bout, while the DA activity in the nAcc appears to be involved in the maintenance of responding during the early and middle stages of a self-administration session (see Refs. [2,29] for more on the phases of the selfadministration bout). While the complex interactions within the mesocortical/ mesolimbic system that process the various stimuli that regulate ethanol self-administration is beyond this discussion, the data suggest that the dual DA feedback processes between the mPFC and the nAcc core play an important part in the termination of a drinking bout. Our working hypothesis for the last several years has been that the onset of a self-administration drinking bout is influenced by external environmental stimuli that function as discriminative and conditioned reinforcing stimuli and are processed at the level of the mPFC. The mPFC then sends excitatory input to the nAcc core that influences the release of DA [31] as well as having direct actions on the medium spiny output neurons in the core [32]. This mPFC output can provide information on the initial salience of the environmental stimulus conditions and instigate the processes that control a directed behavioral response (i.e., lever pressing) that has been previously associated with these stimuli [33,34]. It may be this initial input from the mPFC affecting the release of DA that has been measured before the onset of ethanol drinking in this behavioral situation [35]. In addition to the environmental stimuli, internal stimuli that reach the mPFC from the hypothalamus, amygdala, and hippocampus also interact to determine the degree of glutamatergic output to the nAcc [19,36]. This part of the system is therefore testing for the salience of the external environmental stimuli in conjunction with internal generated stimulus states (i.e., the degree of deprivation, anxiety, etc.). This system organization is similar to that described by Kalivas et al. [19] using the concept of a motive state being regulated by the interactions between the mPFC, nACC, and the VTA. It is

also similar to the original conceptualization of this circuit by Mogenson et al. [37]. Continuing with this conceptualization, the mPFC output to this stimulus complex is, in part, channeled towards both the nAcc core and the VTA. While this mPFC input to the nAcc is providing information as to the salience of the external stimuli to the nAcc, it is also affecting the activity within the VTA. As ethanol self-administration commences and then continues, the input from the mPFC to the VTA is coupled with the direct pharmacological stimuli of the ethanol that is known to influence the activity of the VTA DA cells [38]. This ethanol action will increase the DA output to both the mPFC and the nAcc. The release of DA in the nAcc provides for a feed-forward process to maintain the current behavior in relation to the salience of the current multiple-stimulus inputs to the nAcc. However, the release of DA in the mPFC would tend to decrease mPFC output [39], thus reducing the excitatory facilitation of nAcc and VTA cells in a negative feedback function. At some point, the combined DA input to the mPFC and nAcc will be sufficient to alter nAcc activities and shift the rats’ actions to other behaviors. This should occur when the ethanol-related stimuli reach a point that decreases their salience in relation to the environmental and organismic stimuli (i.e., a satiety process). The termination of responding is most likely controlled by a shift in the nAcc output to the ventral pallidum and its projections to the medial thalamus that send additional feedback to the mPFC [36]. Bilateral ibotenic acid lesions of the mPFC in rodents leads to an inability to shift behaviors [40]. As well, excess DA in the nAcc leads to stereotyped behavior, and can extend an ethanol self-administration session well beyond the ‘‘normal’’ limits [17]. Thus, the complex interaction between the DA feedback processes between the mPFC and nAcc becomes a central part in the switching of ongoing behaviors within a given environment. The data from the present experiment, although not able to completely confirm this hypothesis of the interactive role of the mPFC and nAcc DA feedback processes, support this interpretation of the mechanism for how this complex system may function to initiate and terminate (regulate) a self-administration bout. In addition, our recent findings that the DA D2 antagonist raclopride microinjected into the nAcc not only impacts consummatory behavior but also has a major impact upon ethanol-seeking behavior [11] support the hypothesis that the integration of information within this mPFC – nAcc – VTA circuit is involved in the determination of the salience of external and internal stimuli and the organization of behavior that has been associated with these stimuli in the past. The use of the dual-site microinjection procedure employed in the current study allows for the complex interactions of this system to be examined. When coupled with various models of ethanol consummatory behavior, a more complete understanding of the mechanisms by which the central nervous system regulates ethanol consumption can be obtained.

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Acknowledgements This work was supported by grants AA06845, AA00142, and AA11997 to HHS from the NIAAA. The authors thank Dr. Cristine Czachowski for her comments on the manuscript.

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