Jamie E. Toalston

Ethanol Is Self-Administered Into the Nucleus Accumbens Shell, But Not the Core: Evidence of Genetic Sensitivity

BACKGROUND A previous study indicated that selectively bred alcohol-preferring (P) rats self-administered ethanol (EtOH) directly into the posterior ventral tegmental area at lower concentrations than Wistar rats. The present study was undertaken to determine involvement of the nucleus accumbens (Acb) with EtOH reinforcement, and a relationship between genetic selection for high alcohol preference and sensitivity of the Acb to the reinforcing effects of EtOH. METHODS Adult P and Wistar rats were assigned to groups that self-infused 0 to 300 mg% EtOH into the Acb shell (AcbSh) or Acb Core (AcbC). Rats were placed into 2-lever (active and inactive) operant chambers and given EtOH for the first 4 sessions (acquisition), artificial cerebrospinal fluid (aCSF) for sessions 5 and 6 (extinction), and EtOH again in session 7 (reinstatement). Responding on the active lever produced a 100-nl injection of the infusate. RESULTS Alcohol-preferring rats self-infused 75 to 300 mg% EtOH, whereas Wistar rats reliably self-infused 100 and 300 mg% EtOH into the AcbSh. Both P and Wistar rats reduced responding on the active lever when aCSF was substituted for EtOH, and reinstated responding in session 7 when EtOH was restored. EtOH was not self-infused into the AcbC by P or Wistar rats. CONCLUSIONS The present results indicate that the AcbSh, but not AcbC, is a neuroanatomical structure that mediates the reinforcing actions of EtOH. The data also suggest that, compared to Wistar rats, the AcbSh of P rats is more sensitive to the reinforcing effects of EtOH.

Modeling binge-like ethanol drinking by peri-adolescent and adult P rats

Alcohol binge-drinking, especially among adolescents and young adults, is a serious public health concern. The present study examined ethanol binge-like drinking by peri-adolescent [postnatal days (PNDs 30-72)] and adult (PNDs 90-132) alcohol-preferring (P) rats with a drinking-in-the-dark-multiple-scheduled-access (DID-MSA) procedure used by our laboratory. Male and female P rats were provided concurrent access to 15% and 30% ethanol for three 1-h sessions across the dark cycle 5 days/week. For the 1st week, adolescent and adult female P rats consumed 3.4 and 1.6g/kg of ethanol, respectively, during the 1st hour of access, whereas for male rats the values were 3.5 and 1.1g/kg of ethanol, respectively. Adult intakes increased to ~2.0 g/kg/h and adolescent intakes decreased to ~2.5 g/kg/h across the 6 weeks of ethanol access. The daily ethanol intake of adult DID-MSA rats approximated or modestly exceeded that seen in continuous access (CA) rats or the selection criterion for P rats (≥5 g/kg/day). However, in general, the daily ethanol intake of DID-MSA peri-adolescent rats significantly exceeded that of their CA counterparts. BELs were assessed at 15-min intervals across the 3rd hour of access during the 4th week. Ethanol intake was 1.7 g/kg vs. 2.7 g/kg and BELs were 57 mg% vs. 100mg% at 15- and 60-min, respectively. Intoxication induced by DID-MSA in female P rats was assessed during the 1st vs. 4th week of ethanol access. Level of impairment did not differ between the 2 weeks (106 vs. 97 s latency to fall, 120 s criterion) and was significant (vs. naïve controls) only during the 4th week. Overall, these findings support the use of the DID-MSA procedure in rats, and underscore the presence of age- and sex-dependent effects mediating ethanol binge-like drinking in P rats.

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

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 D2,3 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.

Serotonin-3 receptors in the posterior ventral tegmental area regulate ethanol self-administration of alcohol-preferring (P) rats

Several studies indicated the involvement of serotonin-3 ([5-hydroxy tryptamine] 5-HT(3)) receptors in regulating alcohol-drinking behavior. The objective of this study was to determine the involvement of 5-HT(3) receptors within the ventral tegmental area (VTA) in regulating ethanol self-administration by alcohol-preferring (P) rats. Standard two-lever operant chambers (Coulbourn Instruments, Allentown, PA) were used to examine the effects of seven consecutive bilateral microinfusions of ICS 205-930 (ICS), a 5-HT(3) receptor antagonist, directly into the posterior VTA on the acquisition and maintenance of 15% (vol/vol) ethanol self-administration. P rats readily acquired ethanol self-administration by the fourth session. The three highest doses (0.125, 0.25, and 1.25 microg) of ICS prevented acquisition of ethanol self-administration. During the acquisition postinjection period, all rats treated with ICS demonstrated higher responding on the ethanol lever, with the highest dose producing the greatest effect. In contrast, during the maintenance phase, the three highest doses (0.75, 1.0, and 1.25 microg) of ICS significantly increased responding on the ethanol lever; after the 7-day dosing regimen, responding on the ethanol lever returned to control levels. Microinfusion of ICS into the posterior VTA did not alter the low responding on the water lever and did not alter saccharin (0.0125% wt/v) self-administration. Microinfusion of ICS into the anterior VTA did not alter ethanol self-administration. Overall, the results of this study suggest that 5-HT(3) receptors in the posterior VTA of the P rat may be involved in regulating ethanol self-administration. In addition, chronic operant ethanol self-administration and/or repeated treatments with a 5-HT(3) receptor antagonist may alter neuronal circuitry within the posterior VTA.

The posterior ventral tegmental area mediates alcohol-seeking behavior in alcohol-preferring rats.

The mesolimbic dopamine (DA) system is involved in the rewarding process of drugs of abuse and is activated during the anticipation of drug availability. However, the neurocircuitry that regulates ethanol (EtOH)-seeking has not been adequately investigated. The objectives of the present study were to determine 1) whether the posterior ventral tegmental area (p-VTA) mediates EtOH-seeking, 2) whether microinjections of EtOH into the p-VTA could stimulate EtOH-seeking, and (3) the involvement of p-VTA DA neurons in EtOH-seeking. Alcohol-preferring rats were trained to self-administer 15% EtOH and water. After 10 weeks, rats underwent extinction training, followed by 2 weeks in their home cages. During the home-cage period, rats were then bilaterally implanted with guide cannulae aimed at the p-VTA or anterior ventral tegmental area (a-VTA). EtOH-seeking was assessed by the Pavlovian spontaneous recovery model. Separate experiments examined the effects of: 1) microinjection of quinpirole into the p-VTA, 2) EtOH microinjected into the p-VTA, 3) coadministration of EtOH and quinpirole into the p-VTA, 4) microinjection of quinpirole into the a-VTA, and 5) microinjection of EtOH into the a-VTA. Quinpirole microinjected into the p-VTA reduced EtOH-seeking. Microinjections of EtOH into the p-VTA increased EtOH-seeking. Pretreatment with both quinpirole and EtOH into the p-VTA reduced EtOH-seeking. Microinjections of quinpirole or EtOH into the a-VTA did not alter EtOH-seeking. Overall, the results suggest that the p-VTA is a neuroanatomical substrate mediating alcohol-seeking behavior and that activation of local DA neurons is involved.

Selective breeding for high alcohol preference increases the sensitivity of the posterior VTA to the reinforcing effects of nicotine

The rate of codependency for alcohol and nicotine is extremely high. Numerous studies have indicated that there is a common genetic association for alcoholism and nicotine dependency. The current experiments examined whether selective breeding for high alcohol preference in rats may be associated with increased sensitivity of the posterior ventral tegmental area (pVTA) to the reinforcing properties of nicotine. In addition, nicotine can directly bind to the serotonin‐3 (5‐HT3) receptor, which has been shown to mediate the reinforcing properties of other drugs of abuse within the pVTA Wistar rats were assigned to groups that were allowed to self‐infuse 0, 10, 50, 100, 200, 400 or 800 μM nicotine in two‐lever (active and inactive) operant chambers. P rats were allowed to self‐infuse 0, 1, 10, 50 or 100 μM nicotine. Co‐infusion of 5‐HT3 receptor antagonists with nicotine into the pVTA was also determined. P rats self‐infused nicotine at lower concentrations than required to support self‐administration in Wistar rats. In addition, P rats received more self‐infusions of 50 and 100 μM nicotine than Wistar rats; including a 5HT3 receptor antagonist (LY‐278,584 or zacopride) with nicotine reduced responding on the active lever. Overall, the data support an association between selective breeding for high alcohol preference and increased sensitivity of the pVTA to the reinforcing properties of nicotine. In addition, the data suggest that activation of 5HT3 receptors may be required to maintain the local reinforcing actions of nicotine within the pVTA.

Alcohol-preferring (P) rats are more sensitive than Wistar rats to the reinforcing effects of cocaine self-administered directly into the nucleus accumbens shell

Wistar rats will self-administer cocaine directly into the nucleus accumbens shell (AcbSh), but not into the nucleus accumbens core. In human and animal literature, there is a genetic association between alcoholism and cocaine dependency. The current experiment examined whether selective breeding for high alcohol preference is also associated with greater sensitivity of the AcbSh to the reinforcing properties of cocaine. P and Wistar rats were given cocaine (0, 100, 200, 400, or 800 pmol/100 nl) to self-infuse into the AcbSh. Rats were given cocaine for the first 4 sessions (acquisition), artificial CSF for sessions 5 and 6 (extinction), and cocaine again in session 7 (reinstatement). During acquisition, P rats self-infused 200-800 pmol cocaine (59 infusions/session), whereas Wistar rats only reliably self-infused 800 pmol cocaine (38 infusions/session). Furthermore, P rats received a greater number of cocaine infusions in the 200, 400 and 800 pmol cocaine groups compared to respective Wistar groups during acquisition. Both P and Wistar rats reduced responding on the active lever when aCSF was substituted for cocaine, and reinstated responding in session 7 when cocaine was restored. However, P rats had significantly greater infusions during session 7 compared to session 4 at all concentrations of cocaine tested, whereas Wistar rats only displayed greater infusions during session 7 compared to session 4 at the 400 and 800 pmol cocaine concentrations. The present results suggest that, compared to Wistar rats, the AcbSh of P rats was more sensitive to the reinforcing effects of cocaine. The reinstatement data suggest that the AcbSh of P rats may have become sensitized to the reinforcing effects of cocaine. Overall, the findings from this study support a genetic association between high alcohol preference and greater sensitivity to the reinforcing effects of cocaine.

Effects of ceftriaxone on ethanol, nicotine or sucrose intake by alcohol-preferring (P) rats and its association with GLT-1 expression

Increased glutamatergic neurotransmission appears to mediate the reinforcing properties of drugs of abuse, including ethanol (EtOH). We have shown that administration of ceftriaxone (CEF), a β-lactam antibiotic, reduced EtOH intake and increased glutamate transporter 1 (GLT-1) expression in mesocorticolimbic regions of male and female alcohol-preferring (P) rats. In the present study, we tested whether CEF administration would reduce nicotine (NIC) and/or EtOH intake by adult female P rats. P rats were randomly assigned to 4 groups: (a) 5% sucrose (SUC) or 10% SUC [SUC], (b) 5% SUC+0.07mg/ml NIC and 10% SUC+0.14mg/ml NIC [NIC-SUC], 15% EtOH and 30% EtOH [EtOH] and (d) 15% EtOH+0.07mg/ml NIC and 30% EtOH+0.14mg/ml NIC [NIC-EtOH]. After achieving stable intakes (4weeks), the rats were administered 7 consecutive, daily i.p. injections of either saline or 200mg/kg CEF. The effects of CEF on intake were significant but differed across the reinforcers; such that ml/kg/day SUC was reduced by ∼30%, mg/kg/day NIC was reduced by ∼70% in the NIC-SUC group and ∼40% in the EtOH-NIC group, whereas g/kg/day EtOH was reduced by ∼40% in both the EtOH and EtOH-NIC group. The effects of CEF on GLT-1 expression were also studied. We found that CEF significantly increased GLT-1 expression in the prefrontal cortex and the nucleus accumbens of the NIC and NIC-EtOH rats as compared to NIC and NIC-EtOH saline-treated rats. These findings provide further support for GLT-1-associated mechanisms in EtOH and/or NIC abuse. The present results along with previous reports of CEFs efficacy in reducing cocaine self-administration in rats suggest that modulation of GLT-1 expression and/or activity is an important pharmacological target for treating polysubstance abuse and dependence.

Synergistic self-administration of ethanol and cocaine directly into the posterior ventral tegmental area: involvement of serotonin-3 receptors.

Ethanol (EtOH) and cocaine are both self-administered into the posterior ventral tegmental area (VTA). Self-administration of either drug is prevented by coadministration of a serotonin (5-HT3) receptor antagonist. Electrophysiological studies indicated that cocaine and EtOH can act synergistically to stimulate VTA dopamine neurons. The current experiment assessed whether cocaine and EtOH would synergistically interact to produce a reinforcing action within the posterior VTA. Adult female Wistar rats were randomly assigned to one of 13 groups. There were three control groups: artificial cerebrospinal fluid (aCSF), a subthreshold EtOH (100 mg%) group, and a subthreshold cocaine (25 pmol/100 nl) group. The other groups self-administered 50 or 75 mg% EtOH containing 6.25, 12.5, or 25 pmol/100 nl cocaine or 100 mg% EtOH containing 3.12, 6.25, 12.5, or 25 pmol/100 nl cocaine. All rats received the assigned infusate for sessions 1 through 4, aCSF alone in sessions 5 and 6, and the original infusate during session 7. The effects of adding a 5-HT3 receptor antagonist [tropisetron, C17H20N2O2 (ICS 205,930) and C17H22N4O.C4H4O4 (LY278-584)] on coadministration of EtOH and cocaine (75 mg% + 12.5 pmol/100 nl) were determined. Rats failed to self-administer aCSF or the subthreshold concentration of EtOH or cocaine. All three concentrations of EtOH (50, 75, and 100 mg%) combined with cocaine (12.5 and 25 pmol/100 nl) supported self-administration. Adding a 5HT3 receptor antagonist attenuated coadministration of EtOH + cocaine. Overall, the data indicate that the reinforcing properties of EtOH and cocaine interacted synergistically within the posterior VTA, and these synergistic effects were mediated, at least in part, by activation of local 5-HT3 receptors.

Effects of alcohol and saccharin deprivations on concurrent ethanol and saccharin operant self-administration by alcohol-preferring (P) rats.

Consumption of sweet solutions has been associated with a reduction in withdrawal symptoms and alcohol craving in humans. The objective of the present study was to determine the effects of ethanol and saccharin (SACC) deprivations on operant oral self-administration. Alcohol-preferring (P) rats were allowed to lever press concurrently self-administer ethanol (15% vol/vol) and SACC (0.0125% g/vol) for 8 weeks. Rats were then maintained on daily operant access (nondeprived), deprived of both fluids (2 weeks), deprived of SACC and given 2 ml of ethanol daily, or deprived of ethanol and given 2 ml of SACC daily. All groups were then given 2 weeks of daily operant access to ethanol and SACC, followed by an identical second deprivation period. P rats responded more for ethanol than SACC. All deprived groups increased responding on the ethanol lever, but not on the SACC lever. Daily consumption of 2 ml ethanol decreased the duration of the alcohol deprivation effect (ADE). Home cage access to 2 ml of SACC also decreased the ADE but to a lesser extent than access to ethanol. A second deprivation period further increased and prolonged the expression of an ADE. These results show ethanol is a more salient reinforcer than SACC. With concurrent access to ethanol and SACC, P rats do not display a saccharin deprivation effect. Depriving P rats of both ethanol and SACC had the most pronounced effect on the magnitude and duration of the ADE, suggesting that there may be some interactions between ethanol and SACC in their CNS reinforcing effects.