Project A:The goal of this study is to better understand how DA receptor subtypes modulate collateral GABA synaptic transmission in the nucleus accimbens (NAc). We use double transgenic mice to express ChR2 in iMSNs or dMSNs and label fluorescently dMSNs. We are thus able to record the collateral GABA transmission between the four combinations of MSN subpopulations. Bath application of the D2-like agonist quinpirole inhibits iMSNdMSN and iMSNiMSN GABA transmission to a similar degree. Interestingly, application of D2-like antagonist sulpiride while washing out quinpirole only partially reverses the inhibition at iMSNdMSN synapses, while it fully reverses the inhibition at iMSNiMSN synapses. Thus, quinpirole inhibition is slightly more potent and more reversible at homotypical synapses from iMSNs to iMSNs. Quinpirole has no effect on dMSNdMSN transmission and only a slight effect at dMSNiMSN transmission, consistent with data from the dorsal striatum (Tecuapetla et al., 2009) that suggests DAs effects on synaptic transmission in the striatum are mainly presynaptic in neurons containing D2Rs. These results suggest D2 receptor activation in the NAc mainly acts presynaptically to depress GABA transmission from iMSNs (a subset of collaterals) and that this presynaptic D2 mediated inhibition of iMSN collaterals is similar regardless of postsynaptic MSN target. We next probed the effects of DA, the endogenous agonist, on synaptic transmission in the NAc in order to better understand how these local collateral synapses might be modulated in vivo where D1 and D2 receptors could be coactivated. To our surprise, we found DA produces a more potent inhibition at these synapses compared to D2-like agonists. High concentration DA inhibits iMSNdMSN transmission almost completely. This inhibition is not blocked by pretreatment with D1-like antagonist and is only partially reduced by D2-like antagonist sulpiride. The partial blockade by D2-like antagonist can be mimicked by the cell-specific genetic deletion of D2Rs from iMSNs, which strongly suggests that only a part of the DA mediated inhibition is carried out by D2Rs and the rest seems insensitive to D1-like or D2-like antagonists. Concentration-response curves of the DA mediated inhibition in both the Adora2a-cre+/- and iMSND2KO mice show that removal of D2Rs produces a rightward shift in the dose-response curve, with an 10 fold change in the IC50 and making DA less potent at inhibiting GABA transmission. These results suggest DA is inhibiting iMSNdMSN transmission through both D2 receptor activation as well as an additional heretofore unknown mechanism. The D2R independent mechanism of the DA inhibition appears to be also mediated by a presynaptic receptor/mechanism as DA has no effect on the amplitude of GABA-A mediated currents produced by uncaging of Rubi-GABA. Further, DA increases the paired-pulse ratio at synapses from iMSND2KO. A long list of pharmacological agents has been used to screen the identity of the D2R-independent modulation of GABA release by DA. These include antagonists for the following receptors: D1-like, alpha-adrenergic, muscarinic, 5HT1b/d, 5HT2a/c, adenosine A1, opioid, GABA-B, and CB1 antagonists. Each had no effect on the DA mediated inhibition in the iMSND2KO mice. Project B: Role for dopamine D2 receptors in the stimulatory and depressive effects of alcohol Alcohol produces both stimulant and sedating effects in humans. Clinical literature suggests that people who experience the stimulant effects of alcohol more intensely are more likely to abuse the substance and develop dependency. In animal studies, stimulant and depressive effects of alcohol can be quantified by locomotive activity. Previous research indicates a role for dopamine receptors in the locomotive effects of alcohol, however, a detailed understanding of the receptor class and localization is unknown. The current study examines the role of specific subpopulations of dopamine D2 receptors in modulating the stimulant and depressant effects of alcohol. Using genetically engineered mice lacking D2 receptors on medium spiny neurons (MSNs) in the striatum or D2 autoreceptors on midbrain dopamine neurons, we examined dose-dependent alcohol-induced locomotion. Compared with littermate controls, MSN D2 knockout mice show a significantly increased locomotor response to alcohol, while the mice lacking D2 autoreceptors are more sensitive to the depressive effects of alcohol. To assess differences in alcohol-induced sedation in each of the mouse lines, we performed the Loss of Righting Reflex (LORR). LORR data show that MSN D2 knockout mice are more resilient to the sedative effects of alcohol, with only 50% of mice losing the righting reflex. Meanwhile, D2 autoreceptor knockout mice lose and regained the righting reflex after a high dose of alcohol similar to control mice. To further explore the rewarding and reinforcing aspects of alcohol in these transgenic mice, we examined intake parameters. MSN D2 knockout mice showed ahigher preference for alcohol than controls in a two-bottle choice test and increased seeking in a self-administration paradigm. MSN D2 knockout mice show resilience to quinine adulteration of alcohol compared to controls. These results suggest that MSN striatal dopamine D2 receptors may be playing an important role in modulating the behavioral responses to alcohol. This may provide an explanation for the variation in individuals responses to the stimulant effects of alcohol and the resulting susceptibility to abuse and dependence. Project C: Lack of LRRK2, a Parkinsons disease-related protein, promotes compulsive-like and high alcohol intake in mice. High alcohol drinking and consumption despite aversive consequences is a characteristic of alcohol use disorders. The progression from controlled alcohol use to more compulsive drinking is influenced by many factors, such as psychological, environmental and genetic factors. In order to elucidate the genetic components implicated in the development of the compulsive alcohol consumption, we evaluated changes in the whole striatal transcriptome using a murine model of chronic and free choice alcohol intake. In this model, 60 outbred mice had access to alcohol in a three-bottle choice way during four stages: Acquisition (10 weeks), Withdraw (2 weeks), Re-exposure (2 weeks) and Adulteration (2 weeks, access to alcohol solutions adulterated with bitter tasting quinine). After treatment, animals were classified according to their individual alcohol intake as Light drinkers (preference for water throughout the experiment), Heavy drinkers (preference for alcohol, but significantly intake reduction after the alcohol taste-adulteration) and Inflexible drinkers (preference for alcohol even after the taste-adulteration). In the transcriptome analysis, we found that the expression of Lrrk2 gene was increased exclusively in those mice that were Inflexible drinkers. The Lrrk2 gene produces an AKAP protein which regulates the PKA availability in medium spiny neurons and it is responsible for controlling several neuronal functions. Further validations using the full knockout mice for the Lrrk2 gene (Lrrk2-KO) showed that those animals have an enhanced alcohol preference and consumption when compared with the wild types. Also, Lrrk2-KO mice achieved a higher breakpoint in a progressive ratio schedule, indicating an increased motivation to both alcohol seeking and taking. Compulsive-like behavior were accessed by measuring preference for 3 weeks and by alcohol adulteration test. Once again, Lrrk2-KO mice showed high alcohol intake despite the taste-adulteration, suggesting enhanced compulsive-like alcohol consumption in mice lacking the Lrrk2 gene. Furthermore, the Lrrk2-KO mice showed an increased basal anxiety-like behavior, spending a shorter time on the cen
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