The Edinger-Westphal-lateral septum urocortin pathway and its relationship to alcohol consumption

Identifying and characterizing brain regions regulating alcohol consumption is beneficial for understanding the mechanisms of alcoholism. To this aim, we first identified brain regions changing in expression of the inducible transcription factor c-Fos in the alcohol-preferring C57BL/6J (B6) and alcohol-avoiding DBA/2J (D2) mice after ethanol consumption. Drinking a 5% ethanol/10% sucrose solution in a 30 min limited access procedure led to induction of c-Fos immunoreactivity in urocortin (Ucn)-positive cells of the Edinger-Westphal nucleus (EW), suppression of c-Fos immunoreactivity in the dorsal portion of the lateral septum (LS) of both strains of mice, and strain-specific suppression in the intermediate portion of the LS and the CA3 hippocampal region. Because the EW sends Ucn projections to the LS, and B6 and D2 mice differ dramatically in EW Ucn expression, we further analyzed the Ucn EW–LS pathway using several genetic approaches. We find that D2 mice have higher numbers of Ucn-immunoreactive processes than B6 mice in the LS and that consumption of ethanol/sucrose in the F2 offspring of a B6D2 intercross positively correlates with Ucn immunoreactivity in the EW and negatively correlates with Ucn immunoreactivity in the LS. In agreement with these findings, we find that alcohol-avoiding male B6.D2Alcp1 line 2.2 congenic mice have lower Ucn immunoreactivity in the EW than male B6.B6 mice. Finally, we also find that HAP mice, selectively bred for high alcohol preference, have higher Ucn immunoreactivity in EW, than LAP mice, selectively bred for low alcohol preference. Taken together, these studies provide substantial evidence for involvement of the EW–LS Ucn pathway in alcohol consumption

Initial D₂ Dopamine Receptor Sensitivity Predicts Cocaine Sensitivity and Reward in Rats

<div><p>The activation of dopamine receptors within the mesolimbic dopamine system is known to be involved in the initiation and maintenance of cocaine use. Expression of the D₂ dopamine receptor subtype has been implicated as both a predisposing factor and consequence of chronic cocaine use. It is unclear whether there is a predictive relationship between D₂ dopamine receptor function and cocaine sensitivity that would enable cocaine abuse. Therefore, we exploited individual differences in behavioral responses to D₂ dopamine receptor stimulation to test its relationship with cocaine-mediated behaviors. Outbred, male Sprague-Dawley rats were initially characterized by their locomotor responsiveness to the D₂ dopamine receptor agonist, quinpirole, in a within-session ascending dose-response regimen (0, 0.1, 0.3 & 1.0 mg/kg, sc). Rats were classified as high or low quinpirole responders (HD₂ and LD₂, respectively) by a median split of their quinpirole-induced locomotor activity. Rats were subsequently tested for differences in the psychostimulant effects of cocaine by measuring changes in cocaine-induced locomotor activity (5 and 15 mg/kg, ip). Rats were also tested for differences in the development of conditioned place preference to a low dose of cocaine (7.5 mg/kg, ip) that does not reliably produce a cocaine conditioned place preference. Finally, rats were tested for acquisition of cocaine self-administration and maintenance responding on fixed ratio 1 and 5 schedules of reinforcement, respectively. Results demonstrate that HD₂ rats have enhanced sensitivity to the locomotor stimulating properties of cocaine, display greater cocaine conditioned place preference, and self-administer more cocaine compared to LD₂ animals. These findings suggest that individual differences in D₂ dopamine receptor sensitivity may be predictive of cocaine sensitivity and reward.</p></div

Initial cocaine sensitivity corresponds with differences in D₂ DA receptor sensitivity.

<p>The area under the curve (AUC) was calculated for each rat’s cocaine-induced locomotor activity across both 5 and 15 mg/kg doses. Using this calculated score for initial cocaine-induced locomotor activity, rats were re-classified into a low cocaine responder group (LCR) and a high cocaine responder group (HCR). (A) HCR rats displayed significantly greater quinpirole-induced locomotor activity at the 0.3 and 1.0 mg/kg doses. *HCR significant from LCR, p<0.05. (B) An analysis of the entire cohort was conducted to determine the relationship between quinpirole AUC scores and cocaine AUC scores. A significant positive relationship was identified between initial quinpirole sensitivity and initial cocaine sensitivity.</p

Distributions and averages of quinpirole-induced locomotor activity for LD₂ and HD₂ groups.

<p>(A) Group distributions of the calculated quinpirole area under the curve (AUC) scores used to classify rats into the LD₂ and HD₂ groups. The dotted line represents the median score (<i>M</i> = 15460). (B) Group averages (± sem) of the quinpirole AUC score used to generate the LD₂ and HD₂ groups. The dotted line represents the median score (<i>M</i> = 15460). (C) Distribution of locomotor activity scores (beam breaks/hr) during the ascending within-session quinpirole dose response testing within the LD₂ (gray circles) and HD₂ (red circles) groups. (D) Group averages (± sem) of the quinpirole dose response curve for the LD₂ and HD₂ groups.</p

Lesions of the Edinger–Westphal nucleus in C57BL/6J mice disrupt ethanol-induced hypothermia and ethanol consumption

Abstract The Edinger-Westphal nucleus (EW) is a brain region that has recently been implicated as an important novel neural target for ethanol. Thus, the EW is the only brain region consistently showing elevated c-Fos expression following both voluntary and involuntary ethanol administration. Ethanol-induced c-Fos expression in the EW has been shown to occur in urocortin I-positive neurons. Moreover, previous reports using several genetic models have demonstrated that differences in the EW urocortin I system are correlated with ethanol-mediated behaviours such as ethanol-induced hypothermia and ethanol consumption. The aim of this study was to confirm these relationships using a more direct strategy. Thus, ethanol responses were measured following electrolytic lesions of the EW in male C57BL ⁄ 6J mice. Both EW-lesioned and sham-operated animals were tested for several ethanol sensitivity measures and ethanol consumption in a two-bottle choice test. The results show that lesions of the EW significantly disrupted ethanol-induced hypothermia, while having no effect on pupillary dilation, locomotor activity or ethanol-induced sedation. In addition, EW-lesioned animals showed significantly lower ethanol preference and total ethanol dose consumed in the two-bottle choice test. EW-lesioned animals also consumed less sucrose than sham-operated animals, but did not have altered preferences for sucrose or quinine in a two-bottle choice test. These data support previously observed genetic correlations between EW urocortin I expression and both ethanol-induced hypothermia and ethanol consumption. Taken together, the findings suggest that the EW may function as a sensor for ethanol, which can influence ethanol consumption and preference

HD₂ animals self-administer more cocaine than LD₂ animals.

<p>(A) There were no group differences in the acquisition of an operant response to acquire sucrose pellets. (B) There were significant group differences in the number of cocaine infusions delivered on both a fixed ratio 1 and fixed ratio 5 schedule of reinforcement. ^#significant trend between HD₂ and LD₂ groups, p = 0.08, *HD₂ significant from LD₂, p<0.05.</p

Cocaine self-administration enhances D₂ DA receptor sensitivity in both LD₂ and HD₂ rats.

<p>(A) Quinpirole AUC scores were enhanced across the entire cohort of animals tested following cocaine self-administration. *After cocaine significant from Before cocaine, p<0.05 (B) Likewise, this enhancement was observed across all quinpirole doses. *After cocaine significant from Before cocaine, p<0.05. (C and D) Cocaine-induced enhancements in D₂ DA receptor sensitivity were apparent in both the LD₂ and HD₂ groups using both the quinpirole AUC scores and raw locomotor scores across the quinpirole dose response curve. *After cocaine significant from Before cocaine, p<0.05. Interestingly, the group differences persisted even after cocaine exposure. † HD₂ significant from LD₂, p<0.05.</p

Quinpirole sensitivity is not associated with novelty-induced locomotor activity.

<p>Assessing novelty-induced locomotion during the habituation phase of testing revealed no significant differences between the LD₂ and HD₂ groups. (A) Distribution of novelty-induced locomotor activity scores over the 2-hr testing period. (B) Time course depicting novelty-induced locomotor activity between the LD₂ and HD₂ groups. Animals from this cohort were re-classified into a low responder group (LR) and high responder group (HR) based on their novelty-induced locomotor activity. (C) LR and HR rats did not predict differences in locomotor activity across the quinpirole dose response testing. (D) HR rats displayed significantly greater cocaine-induced locomotor activity across both cocaine doses. *HR significant from LR, p<0.05.</p

HD₂ animals display greater sensitivity to cocaine-induced locomotor activity.

<p>(A) Rats were tested across two cocaine doses (5 and 15 mg/kg, ip) in a within-session procedure. HD₂ animals displayed significantly greater cocaine-induced locomotor activity to 15 mg/kg cocaine, but not 5 mg/kg cocaine. *HD₂ significant from LD₂, p<0.05 (B and C) Analyses of the entire cohort were conducted to determine the relationship between quinpirole AUC scores and cocaine-induced locomotion. A non-significant positive relationship was identified for cocaine-induced activity at the low dose (B, 5 mg/kg cocaine) and a significant positive relationship was identified for cocaine-induced activity at the high dose (C, 15 mg/kg cocaine).</p

Sucrose and cocaine self-administration data set

<p>This data set includes raw data for sucrose intake and cocaine self-administration experiments for all three rat strains used in our study – Trpc4-KO, wild-type and heterozygous animals. The sucrose intake data is over a 10-day period, while the cocaine self-administration data is 13 days. Both data sets include averages for the first 3 days and last three days of experiments, also shown in Figure 2. Catheter patency tests and lever press data is also included. Catheter patency was tested and recorded daily. Lever press data shows the total lever presses (inactive and active) for each animal, which will differ from number of drug infusions due to the time-out period between infusions.</p