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BEHAVIORAL PHARMACOLOGY

The Reinforcing Actions of a Serotonin-3 Receptor Agonist within the Ventral Tegmental Area: Evidence for Subregional and Genetic Differences and Involvement of Dopamine Neurons

Department of Psychiatry, Institute of Psychiatric Research, Indiana University School of Medicine, Indianapolis, Indiana (Z.A.R., V.E.G., D.J., R.L.B., W.J.M.), and Department of Psychology, Purdue School of Science, Indiana University-Purdue University at Indianapolis, Indianapolis, Indiana (J.E.T., S.M.O.).

Received August 17, 2006; accepted February 22, 2007.

	Abstract

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Studies from our laboratory indicated that local perfusion of the ventral tegmental area (VTA) with a serotonin-3 (5-HT₃) receptor agonist increased dopamine (DA) neuronal activity and that the self-infusion of ethanol (EtOH) and cocaine into the posterior VTA could be inhibited with coadministration of a 5-HT₃ receptor antagonist. The study tested the hypothesis that activating 5-HT₃ receptors within the VTA produces reinforcing effects. The study also examined whether there were differences between Wistar rats and a line of rats selectively bred for high alcohol consumption with regard to the self-infusion of a 5-HT₃ receptor agonist within the VTA. Adult female alcohol-preferring (P) and Wistar rats were allowed to self-infuse the 5-HT₃ receptor agonist 1-(m-chlorophenyl)-biguanide (CPBG) into the posterior or anterior VTA. Furthermore, experiments examined the effects of coinfusion of the 5-HT₃ antagonist ICS 205,930 (ICS), and the DA D_2,3 agonist quinpirole on the self-infusion of CPBG. Both Wistar and P rats readily self-administered CPBG into the posterior, but not anterior, VTA. P rats self-infused lower concentrations of CPBG (0.10 µM) than did Wistar rats (1.0 µM). Coinfusion of either ICS or quinpirole reduced CPBG self-infusion into the posterior VTA. The results of this study suggest that activation of 5-HT₃ receptors within the posterior VTA produces reinforcing effects and that these reinforcing effects are mediated through activation of DA neurons. Furthermore, the data suggest that selective breeding for alcohol-preference results in the posterior VTA being more sensitive to the reinforcing effects of CPBG.

The results of several studies suggested a role for the involvement of 5-HT₃ receptors in mediating the excitatory actions of ethanol (EtOH). EtOH can enhance 5-HT₃ receptor-mediated ion currents (Lovinger and White 1991); 5-HT₃ receptor antagonists inhibited the increase in extracellular DA levels in the nucleus accumbens elicited by EtOH when the antagonist was given systemically (Carboni et al., 1989; Wozniak et al., 1990) or locally (Campbell and McBride, 1995). Moreover, the in vivo microdialysis study of Campbell et al. (1996) indicated that local perfusion with a 5-HT₃ receptor antagonist blocked EtOH-stimulated somatodendritic DA release within the VTA, suggesting that the activating effects of EtOH on VTA DA neurons were mediated in part through 5-HT₃ receptors.

The intracranial self-administration (ICSA) technique (Bozarth and Wise, 1980; Goeders and Smith, 1987; Wise and Hoffman, 1992; McBride et al., 1999) has been used to study the reinforcing effects of EtOH within the VTA (Gatto et al., 1994; Rodd-Henricks et al., 2000; Rodd et al., 2004a,b, 2005b). The results of these studies suggested that selective breeding for alcohol preference might increase the sensitivity of the VTA to the reinforcing effects of EtOH (Gatto et al., 1994; Rodd et al., 2004a) and that the posterior VTA supported the self-infusions of EtOH, whereas the anterior VTA did not (Rodd-Henricks et al., 2000; Rodd et al., 2005b). In addition, studies using coinfusion of a D_2,3 agonist (quinpirole) to activate cell body D₂ autoreceptors and reduce DA neuronal activity indicated that the self-infusion of EtOH into the posterior VTA required activation of DA neurons (Rodd et al., 2004b, 2005b).

Because of the evidence indicating that local administration of a 5-HT₃ receptor antagonist in the VTA inhibited EtOH-stimulated somatodendritic DA release (Campbell et al., 1996), our laboratory undertook a study to examine the involvement of 5-HT₃ receptors in the reinforcing effects of EtOH within the posterior VTA of Wistar rats (Rodd-Henricks et al., 2003). The results of this study indicated that coinfusion of 5-HT₃ receptor antagonists blocked the self-infusion of EtOH into the posterior VTA, suggesting that activation of 5-HT₃ receptors is involved in mediating the reinforcing effects of EtOH in this region. In a subsequent study, Rodd et al. (2005b) reported that ICS 205,930 (ICS) also reduced the self-infusion of EtOH into the posterior VTA of alcohol-preferring (P) rats but that this antagonist was significantly less effective in the P rat than Wistar rat in reducing EtOH self-infusions. This difference in the effectiveness of the antagonist to reduce EtOH self-infusions between the P and Wistar rats may be a result of differences in the 5-HT₃ receptors and/or local circuits mediating the actions of the 5-HT₃ receptors within the VTA.

Numerous reports have indicated that the VTA is not a homogenous structure. Arnt and Scheel-Kruger (1979) demonstrated functional differences between the anterior and posterior VTA in the locomotor activating effects of GABA_A agonists and antagonists. Ethanol and acetaldehyde are self-administered into the posterior, but not anterior, VTA (Rodd-Henricks et al., 2000, 2002; Rodd et al., 2004b). In mice, research has shown that injections of a GABA_B agonist into the posterior, but not anterior, VTA increased locomotor activity (Boehm et al., 2002). Cocaine was self-infused into the posterior, but not anterior, VTA of Wistar rats, and this self-infusion could be blocked by coinfusion of quinpirole or a 5-HT₃ receptor antagonist (Rodd et al., 2005a). The subregional differences in self-administration of cocaine are probably related to the differences in neuronal circuitry between the anterior and posterior VTA.

Immunohistochemical studies have indicated that the posterior VTA has a greater density of µ-opioid receptors in the posterior than the anterior VTA (Mansour et al., 1995). Overexpression of an -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit (GluR1) in the posterior and anterior VTA produced differential consequences on morphine-conditioned place preference (Carlezon et al., 2000). A recent series of experiments revealed that the posterior and anterior VTA have distinct innervation patterns (posterior VTA heavily innervates the Acb shell) and that altering cAMP-response element-binding protein levels in the anterior and posterior VTA produced contrasting effects on the reinforcing properties of morphine and cocaine (Olson et al., 2005).

Because the findings suggest that activation of 5-HT₃ receptors is involved in mediating the self-infusions of EtOH and cocaine in the posterior VTA of Wistar and P rats, the current study was undertaken to test the hypothesis that activating 5-HT₃ receptors within the posterior VTA will produce reinforcing effects. This hypothesis was tested using the ICSA technique to study the self-infusion of 1-(m-chlorophenyl)-biguanide (CPBG), a 5-HT₃ receptor agonist, into the VTA of Wistar and P rats. CPBG expresses a 100-fold greater affinity for the 5-HT₃ receptors than serotonin (Kilpatrick et al., 1990), and CPBG has no significant effects at other serotonin receptors (Walcourt-Ambakederemo and Winlow, 1995).

	Materials and Methods

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Experimentally naive, female Wistar rats (Harlan, Indianapolis, IN) and P rats from the 51st and 52nd generations weighing 250 to 320 g at time of surgery were used. Female rats were used in the present study because female rats were used in previous studies involving the ICSA of EtOH and female P rats maintain their body weights and head size better than male P rats for more accurate stereotaxic placements (Gatto et al., 1994; Rodd-Henricks et al., 2000; Rodd et al., 2004a, 2005a,b). Rats were double-housed upon arrival and maintained on a 12-h reverse light/dark cycle (lights off at 9:00 AM). Although not systematically studied, the estrus cycle did not seem to have a significant effect on ICSA behavior in the present or previous ICSA studies (Gatto et al., 1994; Ikemoto et al., 1997, 1998; Rodd-Henricks et al., 2000; Rodd et al., 2004a), as indicated by no obvious fluctuations in ICSA behavior in rats given similar doses of the same agent for four or more sessions. Food and water were freely available except during testing.

Animals used in this study 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 and the Guide for the Care and Use of Laboratory Animals of the National Research Council (NIH, 1996). The number of animals indicated for each experiment represents 95% of the total number that underwent surgery; 5% of the animals were not included for analyses, mainly due to the loss of the guide cannula before completion of all experimental sessions. The data for these animals were not used because their injection sites could not be verified.

General Test Condition. While under isoflurane anesthesia, a unilateral 22-gauge guide cannula (Plastics One, Roanoke, VA) was stereotaxically implanted in the right hemisphere of each subject, aimed 1.0 mm above the target region. Coordinates (Paxinos and Watson 1998) for placements into the posterior VTA were 5.4 mm posterior to bregma, 2.1 mm lateral to the midline, and 8.5 mm ventral from the surface of the skull at a 10° angle to the vertical region. Similar coordinates were used for the female P and Wistar rats in this study. Coordinates (Paxinos and Watson 1998) for placements into the anterior VTA were 4.8 mm posterior to bregma, 2.1 mm lateral to the midline, and 8.5 mm ventral from the surface of the skull at a 10° angle to the vertical region. In between experimental sessions, a 28-gauge stylet was inserted into the guide cannula and extended 0.5 mm beyond the tip of the guide. After surgery, all rats were individually housed and allowed to recover 7 to 10 days. Animals were handled for at least 5 min daily after the 4th recovery day. Subjects were not acclimated to the experimental chamber before the commencement of data collection, nor were they trained on any other operant paradigm.

The experimental chambers (30 x 30 x 26 cm; width x height x diameter) were situated in a sound-attenuating cubicle (64 x 60 x 50 cm; Coulbourn Instruments, Allentown, PA) and illuminated by a dim house-light during testing. Two identical levers (3.5 x 1.8 cm) were mounted on a single wall of the experimental chamber, 15 cm above a grid floor, and were separated by 12 cm. Levers were raised to this level to avoid accidental brushing against the lever and to reduce responses as a result of general locomotor activation. Directly above each lever was a row of three different colored cue lights. The light (red) to the far right over the active bar was illuminated during resting conditions. A desktop computer equipped with an operant control system (L2T2 system; Coulbourn Instruments) recorded the data and controlled the delivery of infusate in relation to lever response.

An electrolytic microinfusion transducer system (see Bozarth and Wise, 1980) was used to control microinfusions of drug or vehicle. In brief, two platinum electrodes were placed in an infusate-filled cylinder container (28 mm in length x 6 mm in diameter) equipped with a 28-gauge injection cannula (Plastics One). The electrodes were connected by a spring-coated cable (Plastic One) and a swivel (model 205; Mercotac, Carlsbad, CA) to a constant current generator (MNC, Shreveport, LA) that delivered 6 µA of quiescent current or 200 µA of infusion current between the electrodes. Depression of the active lever delivered the infusion current for 5 s, which led to the rapid generation of H₂ gas (raising the pressure inside the airtight cylinder) and, in turn, forcing 100 nl of the infusate through the injection cannula. During the 5-s infusion and an additional 5-s timeout period, the house light and right cue light (red) were extinguished and the left cue light (green) over the active lever flashed on and off at 0.5-s intervals.

The artificial cerebrospinal fluid (aCSF) consisted of 120.0 mM NaCl, 4.8 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 25.0 mM NaHCO₃, 2.5 mM CaCl₂, and 10.0 mM d-glucose. CPBG, ICS 205,930 (ICS), and quinpirole (all purchased from Sigma, St. Louis, MO) were dissolved in the aCSF solution. When necessary, 0.1 M HCl or 0.1 M NaOH was added to the solutions to adjust pH levels to 7.4 ± 0.1.

For testing, rats were brought to the testing room, the stylet was removed, and the injection cannula was screwed into place. To avoid trapping air at the tip of the injection cannula, the infusion current was delivered for 5 s during insertion of the injector, which resulted in a single noncontingent administration of infusate at the beginning of the session. Injection cannulae extended 1.0 mm beyond the tip of the guide. The experimental chamber was equipped with two levers. Depression of the "active lever" (FR1 schedule of reinforcement) caused the delivery of a 100-nl bolus of infusate over 5 s followed by a 5-s time-out period. During both the 5-s infusion period and 5-s time-out period, responses on the active lever did not produce further infusions. Responses on the "inactive lever" were recorded but did not result in infusions. The assignment of active and inactive levers with respect to the left or right position was counterbalanced among subjects. However, the active and inactive levers remained the same for each rat throughout the experiment. No shaping technique was used to facilitate the acquisition of lever responses. The number of infusions and responses on the active and inactive lever were recorded. The duration of each test session was 4 h, and sessions occurred every other day.

Self-Administration of CPBG into the VTA of Wistar and P Rats. Wistar rats with cannulae placements aimed at the posterior VTA were randomly assigned to one of five groups (n = 42, 7–9/group). A vehicle group received infusions of aCSF for all seven sessions. The other groups were given 0.1, 1, 10, or 50 µM CPBG for the initial four sessions, aCSF for sessions 5 and 6 (extinction), and the original infusate for session 7 (reinstatement). Wistar rats with cannulae placements aimed at the anterior VTA were randomly assigned to one of three groups (n = 24, 7–9/group). A vehicle group received infusions of aCSF for all seven sessions. The other groups were given 10 or 50 µM CPBG for the initial four sessions, aCSF for sessions 5 and 6 (extinction), and the original infusate for session 7 (reinstatement). In addition, there were eight Wistar rats with cannulae placement outside of the VTA. These rats were not included in the statistical analyses.

P rats with cannulae placements aimed at the posterior VTA were randomly assigned to one of seven groups (n = 61, 8 –10/group). A vehicle group received infusions of aCSF for all seven sessions. The other groups were given 0.01, 0.1, 1, 10, 50, or 100 µM CPBG for the initial four sessions, aCSF for sessions 5 and 6 (extinction), and the original infusate for session 7 (reinstatement). P rats with cannulae placements aimed at the anterior VTA were randomly assigned to one of four groups (n = 32, 8/group). A vehicle group received infusions of aCSF for all seven sessions. The other groups were given 1, 10, or 100 µM CPBG for the initial four sessions, aCSF for sessions 5 and 6 (extinction), and the original infusate for session 7 (reinstatement). In addition, there were six P rats with cannulae placement outside of the VTA. These rats were not included in the statistical analyses.

Coinfusion of ICS during Maintenance of CPBG Self-Infusions. Wistar rats were randomly assigned to one of three groups (n = 23, 7–8/group). All groups self-infused 10 µM CPBG during the initial four sessions, 10 µM CPBG and 10, 100, or 200 µM ICS during sessions 5 and 6, and 10 µM CPBG alone during session 7. A control group given ICS alone was not included in the present study because prior studies indicated that ICS alone or other 5-HT₃ receptor antagonists did not impair lever responding. For example, in one study, we examined the effects of coadministration of three 5-HT₃ antagonists on the self-infusion of EtOH into the posterior VTA (Rodd-Henricks et al., 2003). Responses on the active and inactive lever for 100 µM zacopride, LY278-584, or ICS 205,930 were similar to responses on these levers for aCSF self-infusions into the posterior VTA of Wistar rats (Rodd-Henricks et al., 2003), suggesting that these antagonists do not impair operant responding when infused into the posterior VTA. Another ICSA study indicated that higher concentrations of ICS (400 µM) did not affect acetaldehyde self-infusions (>100 active lever responses/session) into the posterior VTA (Rodd et al., 2005b).

Coinfusion of Quinpirole during Maintenance of CPBG Self-Infusion. Wistar rats were randomly assigned to one of three groups (n = 15, 5/group). All groups self-infused 10 µM CPBG during the initial four sessions, 10 µM CPBG and 1, 10, or 100 µM quinpirole during sessions 5 and 6, and 10 µM CPBG alone during session 7. The concentrations of quinpirole used were derived from studies indicating that coadministration of 1 µM did not alter ethanol or acetaldehyde self-administration into the posterior VTA but that 100 µM was effective at reducing self-administration (Rodd et al., 2004b, 2005b). A control group given only quinpirole was not included in the present study because prior studies indicated that quinpirole by itself did not alter operant responding when infused into the posterior VTA (Rodd et al., 2004b, 2005b).

Histology. At the termination of the experiment, 1% bromphenol blue (0.5 µl) was injected into the infusion site. Subsequently, the animals were given a fatal dose of pentobarbital (Nembutal) and then decapitated. Brains were removed and immediately frozen at –70°C. Frozen brains were equilibrated at –15°C in a cryostat microtome and then sliced into 40-µm sections. Sections were then stained with cresyl violet and examined under a light microscope for verification of the injection site using the rat brain atlas of Paxinos and Watson (1998).

Statistical Analysis. Data analysis consisted of a group x day mixed ANOVA, with a repeated measure of "day" (all seven sessions) performed on the number of infusions. Furthermore, for each individual group, lever discrimination was determined by type (active or inactive) x day mixed ANOVA with a repeated measure of day. Lever discrimination is a key factor when a stimulant is self-administered (e.g., EtOH, cocaine, amphetamine). Without a detailed analysis of lever discrimination, it is impossible to distinguish between reinforcement-contingent behavior and drug-stimulated locomotor activity. The direct comparison between P and Wistar rats examined the mean number of infusions received during the first four ICSA sessions with a line x group between subject ANOVAs. To determine the effects of coadministration of ICS or quinpirole on CPBG self-administration, two separate repeated measure ANOVAs were performed. The first ANOVA examined the number of infusions between session 4 (last day of CPBG alone self-administration) and sessions 5 and 6 (coadministration sessions) with group as a between-subject factor. The second ANOVA examined the number of infusions between sessions 5 and 6 (coadministration) and session 7 (CPBG reinstatement), with coadministration group as a between-subject factor.

	Results

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The anterior VTA was defined as the VTA region at the level of the mammillary nuclei, coronal sections –4.8 to –5.2 mm relative to bregma (Fig. 1). The posterior VTA was defined as the VTA region at the level of the interpeduncular nucleus, coronal sections –5.3 to –6.04 mm relative to bregma (Fig. 1). Accidental cannula placements surrounding the VTA included injection sites located in the substantia nigra (three P rats, four Wistar rats), red nucleus (two P rats, two Wistar rats), and caudal linear nucleus of the raphe (one P rat, two Wistar rats). Rats with injector tip placements outside the VTA in both P and Wistar rats displayed an overall low level of infusions and active lever responding throughout all sessions (average infusions and active lever responses for initial four sessions were 8.2 ± 0.7 and 14.6 ± 1.2, respectively). For all sessions, the number of infusions of CPBG for placements outside the VTA was not significantly different from the aCSF group with injection sites in the VTA (p values > 0.58). Likewise, examination of the active lever responses revealed that rats administering CPBG into areas outside the VTA displayed equivalent low levels of responding on both the active and inactive levers (p values > 0.68).

View larger version (64K): [in this window] [in a new window]   Fig. 1. The figure is an illustration of representative injection sites in the anterior and posterior VTA of Wistar (depicted on left side of diagrams) and P (depicted on right side of diagrams) rats. Overlapping sites are not indicated. On the right side of the illustration, for the P rat, closed circles represent injection sites within the posterior VTA (defined as –5.3 to –6.0 mm bregma), and closed squares represent injection sites within the anterior VTA (defined as –4.8 to –5.2 mm bregma). On the left side of the illustration, for the Wistar rat, open circles represent injection sites within the posterior VTA, and open squares indicate injection sites within the anterior VTA.

Self-Infusion of CPBG into the VTA by Wistar and P Rats. A select range of CPBG concentrations infused into the posterior VTA supported self-infusions by Wistar rats. For Wistar rats receiving infusions into the posterior VTA, an ANOVA on the average number of infusions (Fig. 2, left) received during the initial 4 test days (acquisition) revealed a significant effect of dose (F_4,37 = 10.8; p < 0.0001). Post hoc comparisons (Tukey's b) indicated that rats given 1.0, 10, and 50 µM CPBG received significantly more infusions than rats given aCSF. Furthermore, rats given 10 µM CPBG received more infusions than all other groups. In contrast, in the anterior VTA, 10 and 50 µM CPBG were not significantly self-infused above aCSF levels by Wistar rats (Fig. 2, F_2,21 = 0.5; p = 0.61).

View larger version (17K): [in this window] [in a new window]   Fig. 2. Average number of infusions by Wistar rats (±S.E.M.) across the initial four sessions (acquisition) as a function of infusate concentration (0 –50 µM CPBG) and cannula placement (posterior or anterior VTA). Asterisks indicate infusions significantly higher than aCSF. +, a significantly higher value for 10 µM CPBG compared with all other groups (p < 0.05; Tukey's b post hoc; n = 7–9/group).

View larger version (24K): [in this window] [in a new window]   Fig. 3. The number of active and inactive lever presses (means ± S.E.M.) for Wistar rats self-infusing 0, 10, or 50 µM CPBG into the posterior or anterior VTA during sessions 1 to 4, aCSF for sessions 5 and 6, and original infusate during session 7. Asterisks indicate significantly (p < 0.05; Tukey's b) higher responding on the active lever versus responding observed for rats self-administering aCSF and versus responses on the inactive lever (p < 0.05) within a given infusate group (determined by one-way ANOVAs performed on individual sessions contrasting active and inactive lever presses). Plus symbols represent significantly more responding on the active lever compared to all other groups as well as to the inactive lever.

In contrast to the posterior VTA data, Wistar rats given the effective concentrations of CPBG to self-infuse into the anterior VTA (Fig. 3, right) had low levels of responding on the active lever (18 responses/session) that did not differ from responses on the inactive lever or from active lever responses for the aCSF group (20.4 ± 8.2 responses/session).

For P rats with injection sites in the posterior VTA, an ANOVA on the average number of infusions (Fig. 4, left) received during the initial 4 test days (acquisition) revealed a significant effect of dose (F_6,54 = 36.2; p < 0.0001). Post hoc comparisons indicated that P rats given 0.1, 1.0, 10, 50, and 100 µM CPBG received significantly more infusions than rats given only aCSF. Furthermore, P rats given 10 µM CPBG received significantly more infusions than all the other groups, whereas P rats given 0.1, 1.0, 10, and 50 µM CPBG received more infusions than rats given 100 µM CPBG. In contrast, for P rats with placements in the anterior VTA, none of the CPBG concentrations tested was significantly self-infused above aCSF levels (Fig. 4, right; p values > 0.57).

View larger version (19K): [in this window] [in a new window]   Fig. 4. Average number of infusions by P rats (± S.E.M.) across the initial four sessions (acquisition) as a function of infusate concentration (0 –100 µM CPBG) and cannula placement (posterior or anterior VTA). Asterisks indicate infusions significantly higher than aCSF and 0.01 and 100 µM CPBG values. Plus symbol indicates significantly higher value for 10 µM CPBG compared to all other groups, whereas the symbol indicates significantly higher infusions than aCSF (p < 0.05; Tukey's b post hoc; n = 8 –10/group).

P rats self-administering CPBG into the posterior VTA readily discriminated between the active and inactive lever at concentrations ranging from 0.1 to 100 µM CPBG (Fig. 5 shows responses on the active and inactive levers for 0.1, 1.0, and 10 µM CPBG). Furthermore, P rats self-administering 0.01 µM CPBG displayed lever discrimination during sessions 3, 4, and 7 (p values <0.044; data not shown), and received more self-infusions than aCSF during those sessions (individual ANOVAs performed for each session, indicated by post hoc Tukey's b). Responses on the active and inactive levers by P rats self-infusing CPBG with placements in the anterior VTA were comparable to responses when aCSF was given (and to responding by Wistar rats given aCSF in the posterior VTA or CPBG with placements in the anterior VTA) and did not display lever discrimination (p values > 0.41; data not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 5. Number of active and inactive lever presses (means ± S.E.M.) for P rats self-infusing 0.1, 1.0, or 10 µM CPBG into the posterior VTA during sessions 1 to 4, aCSF in sessions 5 and 6, and CPBG again in session 7. Asterisks indicate significantly (p < 0.05) higher responding on the active lever versus responding observed for rats self-administering aCSF and versus responses on the inactive lever within a given infusate group (determined by one-way ANOVAs performed on individual sessions contrasting active and inactive lever presses). Plus symbols indicate significantly more responding than all other groups and responses on the inactive lever.

View larger version (31K): [in this window] [in a new window]   Fig. 6. Patterns of responding on the active lever by Wistar (left) and P (right) rats self-infusing 10 µM CPBG into the posterior VTA. Overall, both the Wistar and P rats readily acquired CPBG self-infusion during the first operant session, displayed a similar pattern of responding during maintenance (session 4), extinguished responding when aCSF alone was given (session 6), and reinstated responding on the active lever when 10 µM CPBG was returned (session 7).

Coinfusion of ICS or Quinpirole on CPBG Self-Infusions. Throughout the four acquisition sessions, rats readily self-infused 10 µM CPBG and responded significantly more on the active than inactive lever (all F_1,6 values > 26.7; all p values < 0.001; Fig. 7). Repeated measure ANOVAs performed on the number of active lever presses and infusions obtained from sessions 4 to 6, with an independent variable of ICS concentration, indicated a significant day x concentration interaction for both dependent variables (F_4,40 > 8.1; p values < 0.0001). Coadministration of 100 or 200 µM ICS in sessions 5 and 6 reduced the number of active lever responses (all F_2,6 values > 47.5; all p values < 0.023), whereas 10 µM ICS did not alter CPBG self-administration (p = 0.87). When CPBG alone was given during session 7, responding on the active lever returned to levels observed in sessions 3 and 4 for the rats that had coinfused 100 or 200 µM ICS. Similar effects were observed for number of infusions/session. In brief, the number of infusions obtained was significantly lower during session 6 compared with session 4 in rats that were coadministered with 100 or 200 µM ICS by 68% (47 infusions reduced to 15) or 63% (45 infusions reduced to 17), respectively. In contrast, for rats that self-infused 10 µM CPBG and 10 µM ICS, there was a nonsignificant increase in the number of infusions obtained during session 6 compared with session 4 (54 compared with 49 infusions, respectively).

View larger version (20K): [in this window] [in a new window]   Fig. 7. Effects of coinfusing ICS 205,930 on the self-infusion of 10 µM CPBG into the posterior VTA by Wistar rats. For the first four sessions, 10 µM CPBG alone was given. In sessions 5 and 6, ICS 205,930 (10, 100, or 200 µM) was coinfused with 10 µM CPBG. In session 7, only 10 µM CPBG was given. Data are the means ± S.E.M.; n = 7–8/group. Asterisks indicate responses on the active lever significantly (p < 0.05) higher than responses on the inactive lever. Plus symbols indicate that responses on the active lever in sessions 5 and 6 are significantly (p < 0.05) lower than responses on the active lever in sessions 4.

View larger version (22K): [in this window] [in a new window]   Fig. 8. Effects of coinfusing quinpirole on the self-infusion of 10 µM CPBG into the posterior VTA by Wistar rats. For the first four sessions, 10 µM CPBG alone was given. In sessions 5 and 6, quinpirole (1, 10, or 100 µM) was coinfused with 10 µM CPBG. In session 7, only 10 µM CPBG was given. Data are the means ± S.E.M.; n = 5/group. Asterisks indicate responses on the active lever significantly (p < 0.05) higher than responses on the inactive lever. Plus symbols indicate that responses on the active lever in sessions 5 and 6 are significantly (p < 0.05) lower than responses on the active lever in session 4.

	Discussion

Top Abstract Materials and Methods Results Discussion References

The reinforcing effects of CPBG could also occur, in part, through activation of 5-HT₃ receptors located on GABA neurons. The inhibition of GABA activity in the VTA, which leads to an increase in DA neuronal activity in the VTA, following cocaine administration is mediated through 5-HT receptors (Cameron and Williams, 1994). Although the interaction between GABA and 5-HT is thought to be primarily mediated through 5-HT_1B/D and 5-HT_2A/C receptors, there is evidence that some effects of activating 5-HT₃ receptors are mediated by GABA neurons (Bonagamba et al., 2000; Bankson and Yamamoto, 2004).

For both the Wistar and P rats, CPBG is self-infused into the posterior VTA but not into the anterior VTA (Figs. 2 and 4). These findings are in agreement with several studies (Ikemoto et al., 1998; Rodd-Henricks et al., 2000; Ikemoto and Wise, 2002; Rodd et al., 2004b, 2005a,b) indicating that the posterior VTA but not the anterior VTA is involved in supporting self-infusion behavior. The differences between the anterior and posterior VTA with regard to the reinforcing effects of these pharmacological agents may be a result of a combination of neuroanatomical factors, such as a) more DA neurons in the posterior than anterior VTA projecting to the nucleus accumbens (Olson et al., 2005), b) a higher proportion of DA to GABA neurons in the posterior than anterior VTA (Olson et al., 2005), c) the posterior VTA having higher 5-HT innervation (Hervé et al., 1987), and d) the VTA containing DA neurons with topographical afferent and efferent projections (Kalén et al., 1988; Brog et al., 1993; Tan et al., 1995) that may differ between the anterior and posterior sites. Furthermore, possible explanations for that lack of self-infusion of CPBG into the anterior VTA are that too few 5-HT₃ receptors and/or the cellular distributions of 5-HT₃ receptors within anterior VTA do not favor activation of DA neurons. These possibilities could also explain why ethanol (Rodd-Henricks et al., 2000) and cocaine (Rodd et al., 2005a) are not self-infused into the anterior VTA, because their reinforcing effects involve activation of 5-HT₃ receptors. In contrast, it is possible that the anterior VTA may not be involved in mediating local chemically induced reinforcing effects. Thus far, limited ICSA studies have suggested that the posterior VTA rather than the anterior VTA is involved in supporting reinforcement processes (Ikemoto et al., 1998; Rodd-Henricks et al., 2000; Ikemoto and Wise, 2002; Rodd et al., 2004b, 2005a,b), although additional studies may indicate involvement of the anterior VTA as well.

The posterior VTA of the P rat seems to be more sensitive and more responsive to the reinforcing effects of CPBG than the posterior VTA of Wistar rats (Figs. 2, 3, 4, 5). The P rats self-infused lower concentrations of CPBG (0.01–0.10 µM) than did Wistar rats (0.10 –1.0 µM) and received significantly more self-infusion than did Wistar rats (e.g., self-infusions for P rats at 1.0 and 10.0 µM CPBG were approximately 40 and 80, whereas the number of self-infusions for the Wistar rats at these two concentrations were approximately 25 and 50). Furthermore, the concentration of 5-HT₃ receptors antagonists required to block EtOH self-administration into the posterior VTA is higher (4-fold) in P rats compared with Wistar rats (Rodd et al., 2004). These results suggest that there may be differences between the P and Wistar rats in the function (e.g., affinity) of the 5-HT₃ receptor, number of 5-HT₃ receptors, cellular distribution of 5-HT₃ receptors, and/or neuronal circuitry within the posterior VTA between P and Wistar rats. Differences between the P and Wistar rats, with regard to the 5-HT₃ receptor, may also reflect differences in the excitatory tone of the VTA DA neuronal system, possibly making the posterior VTA of the P rat generally more sensitive than the posterior VTA of Wistar rats to reinforcing effects of a wide range of compounds. However, studies examining such comparisons between P and Wistar rats have only been completed for EtOH (Rodd et al., 2004a) and CPBG; in both cases, the posterior VTA of the P rat was more sensitive. Because the reinforcing effects of ethanol seem to involve activation of 5-HT₃ receptors, the greater sensitivity of the P rat to the reinforcing effects of ethanol may be related to the differences in the posterior VTA 5-HT₃ receptors (Figs. 2 and 4) between the P and Wistar rats.

The dose response for the self-infusion of CPBG yielded an inverted "U-shape" plot that is most notable in the P rat (because of a more comprehensive dose range; Fig. 4) than Wistar rat (Fig. 2). The inverted U-shape plot may be a result of the higher concentrations giving a greater reinforcing effect with each infusion (therefore, fewer infusions are needed as the dose increases beyond 10 µM CPBG) and/or an effect of the higher concentrations of CPBG acting at sites other than 5-HT₃ receptors. For example, the reduction in self-infusions at the highest concentrations may be a result of CPBG inhibiting DA uptake (Campbell et al., 1995). Inhibiting DA reuptake would lead to increased extracellular levels of DA, which could act at D₂ autoreceptors and reduce DA neuronal activity. Reducing VTA DA neuronal activity would result in lower CPBG self-infusions. The results with quinpirole indicate that activating D₂ autoreceptors within the posterior VTA reduces CPBG self-infusions (Fig. 8), suggesting that the reinforcing effects of CPBG are dependent upon the activity of DA neurons. The observation that the peak number of infusions occurred at 10 µM CPBG for both the P and Wistar rats could reflect the action of CPBG at the DA transporter at the higher concentrations. In contrast, the reduced responding on the active lever at the highest concentrations of CPBG could be a result of nonspecific effects on general motor performance.

Within the first session, both Wistar (Fig. 3) and P (Fig. 5) rats readily learned to discriminate the active lever from the inactive lever when CPBG was self-administered into the posterior but not anterior VTA. Animals used in this study have had no previous experience in the operant chambers. This rapid learning to discriminate the active from the inactive lever within one or two sessions is in agreement with several studies for the self-infusion of EtOH (Gatto et al., 1994; Rodd-Henricks et al., 2000, 2003), cocaine (Rodd et al., 2005a), or opioids (Bozarth and Wise, 1981; Devine and Wise, 1994) into the VTA. An operant oral self-administration study with P rats indicated that it takes four sessions for these rats to learn to discriminate the EtOH-reinforced lever from the water-reinforced lever (Rodd-Henricks et al., 2002) and that Wistar rats typically need to undergo extensive shaping techniques to learn to respond for a reinforcer. The mechanisms underlying the learning processes for readily being able to discriminate the active from the inactive lever within the first session of the ICSA experiments are unknown but are probably related to direct chemical stimulation of VTA DA systems.

The pattern of responding on the active lever (Fig. 6) indicated that, in the first session, both the Wistar and P rats were actively rearing up and pressing the active lever throughout the 4-h session. In the fourth session, the Wistar and P rats seemed to "load up" in the first 30 to 60 min and again in the last 30 to 60 min of the session. During session 6 (the second aCSF session), the pattern of responding was again similar for the Wistar and P rats. The small amount of responding on the active lever that did occur was observed within the first 30 min (Fig. 6); when no reinforcement was received in this initial period, there was almost no responding on the active lever thereafter by either the P or Wistar rat. When CPBG was restored in session 7, high levels of responding on the active lever were evident beyond the initial 30- to 60-min segment, suggesting a "deprivation-like effect", which was more pronounced in the P rat than Wistar rat.

A major concern with the ICSA technique was that diffusion away from the target site may be producing self-infusion behavior. Diffusion was occurring with the present experimental conditions. However, with the small volumes, injected diffusion to sites adjacent to the target site does not seem to contribute to the self-infusion behavior. For one, there is a clear delineation between the effects observed in the present study (Figs. 2 and 4) and in previous studies (Ikemoto et al., 1998; Rodd et al., 2004b, 2005a,b) for self-infusions between the anterior and posterior VTA. In addition, the ICSA procedure did not elicit self-administration behavior when placements were located in sites adjacent to the target region (Ikemoto et al., 1997; Rodd-Henricks et al., 2000). Therefore, with the present experimental conditions, it does not seem that diffusion of CPBG to sites adjacent to the posterior VTA is contributing to self-infusion behavior. In summary, the present findings support the hypothesis that activating 5-HT₃ receptors within the posterior VTA produces reinforcing effects and that these effects are mediated by activation of VTA DA neurons.

	Footnotes

 
This work was supported in part by Research Grants AA10721, AA11261, and AA12262 and by Center Grant AA07611 from National Institute on Alcohol Abuse and Alcoholism.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.112607.

ABBREVIATIONS: 5-HT, serotonin, ICSA, intracranial self-administration; ICS 205,930 (ICS), tropisetron; CPBG, 1-(m-chlorophenyl)-biguanide; P rat, alcohol-preferring rat; EtOH, ethanol; aCSF, artificial cerebrospinal fluid; LY278-584, 1-methyl-N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-1H-indazole-3-carboxamide; ANOVA, analysis of variance; DA, dopamine.

Address correspondence to: Dr. Zachary A. Rodd, Indiana University School of Medicine, Institute of Psychiatric Research, 791 Union Drive, Indianapolis, IN 46202–4887. E-mail: zrodd{at}iupui.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

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