Browsing by Subject "Neurotoxicology"
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Item Achieving pharmacologically relevant IV alcohol self-administration in the rat(2012-09-27) Windisch, Kyle Allyson; Czachowski, Cristine L.; Grahame, Nicholas J.; Kosobud, Ann E. K.Alcohol consumption produces a complex array of effects that can be divided into two types: the explicit pharmacological effects of ethanol (which can be quite separate temporally from time of intake) and the more temporally “relevant” effects (primarily olfactory and taste) that bridge the time from intake to the onset of the pharmacological effects. Dissociating these effects is essential to untangling the neurologic underpinnings of alcohol abuse and dependence. Intravenous self-administration of ethanol allows for controlled and precise dosing, bypasses first order absorption kinetics allowing for a faster onset of pharmacologic effects, and eliminates the confounding “non-pharmacological” effects associated with oral consumption. Intravenous self-administration of ethanol has been reliably demonstrated in both mouse and human experimental models; however, consistent intravenous self-administration of pharmacologically relevant levels of ethanol remains elusive in the rat. Previous work has demonstrated reliable elevated intravenous ethanol self administration using a compound reinforcer of oral sucrose and intravenous ethanol. The present study sought to elucidate the role of each component of this reinforcer complex using a multiple schedule study design. Male P rats had free access to both food and water during all intravenous self-administration sessions and all testing was performed in conjunction with the onset of the dark cycle. Once animals achieved stable operant responding on both levers for an orally delivered 1% sucrose solution (1S) on a FR4 schedule, surgery was conducted to implant an indwelling jugular catheter. Animals were habituated to the attachment of infusion apparatus and received twice daily sessions for four days to condition each lever to its associated schedule. Animals were then trained to respond on a multiple FR4-FR4 schedule composed of alternating 2.5 minute components. During one component only oral 1S was presented, while in the second component a compound reinforcer of oral 1S + IV 20% ethanol was presented (25 mg/kg/injection). Both levers were extended into the chamber during the session, with the active lever/schedule alternating as the session progressed across components. Average ethanol intake was 0.47 ± 0.04 g/kg. A significant increase in sucrose only reinforcers and sucrose lever error responding was found suggesting that sucrose not ethanol is responsible for driving overall responding. The current findings suggest that the existing intravenous ethanol self-administration methodology remains aversive in the rat.Item CaMKII regulation of astrocytic glutamate uptake(2016-05-19) Chawla, Aarti R.; Hudmon, Andy; Cummins, Theodore; Oxford, Gerry S.; Chen, Jinhui; Hoang, QuyenGlutamate clearance by astrocytes is an essential part of physiological excitatory neurotransmission. Failure to adapt or maintain low levels of glutamate in the central nervous system is associated with multiple acute and chronic neurodegenerative diseases. The primary excitatory amino acid transporters (EAATs) in human astrocytes are EAAT1 and EAAT2 (GLAST and GLT-1 respectively in rodents). While the inhibition of a ubiquitously-expressed serine/threonine protein kinase, the calcium/calmodulindependent kinase (CaMKII) results in diminished glutamate uptake in cultured primary rodent astrocytes, the molecular mechanism underlying this regulation is unknown. In order to delineate this mechanism, we use a heterologous expression model to explore CaMKII regulation of EAAT1 and EAAT2. In transiently transfected HEK293T cells, pharmacological inhibition of CaMKII and overexpression of a dominant-negative version of CaMKII (Asp136Asn) reduces [3H]-glutamate uptake by EAAT1, without altering EAAT2 mediated glutamate uptake. Surprisingly, overexpression of a constitutively active autophosphorylation mutant (Thr287Asp) to increase autonomous CaMKII activity and a mutant incapable of autophosphorylation (Thr287Val) had no effect on either EAAT1 or EAAT2 mediated glutamate uptake. Pulldown of FLAGtagged glutamate transporters suggests CaMKII does not interact with EAAT1 or EAAT2. SPOTS peptide arrays and recombinant GST-fusion proteins of the intracellular N- and C-termini of EAAT1 identified two potential phosphorylation sites at residues Thr26 and Thr37 in the N-terminus. Introducing an Ala (a non-phospho mimetic) but not an Asp (phosphomimetic) at Thr37 diminished EAAT1-mediated glutamate uptake, suggesting that the phosphorylation state of this residue is important for constitutive EAAT1 function. In sum, this is the first report of a glutamate transporter being identified as a direct CaMKII substrate. These findings indicate that CaMKII signaling is a critical driver of homeostatic glutamate uptake by EAAT1. Aberrations in basal CaMKII activity disrupt glutamate uptake, which can perpetuate glutamate-mediated excitotoxicity and result in cellular death.Item Characterization of Behavioral Profiles for Inbred P and NP and Congenic P.NP and NP.P Rats(2012-08-27) Jensen, Meredith; Grahame, Nicholas J.; Stewart, Robert; Czachowski, Cristine; Roman, ErikaAlcoholism inheritance rates have been estimated as high as 60% in a human population. Many significant features of alcohol dependence have been replicated in rodent animal models of alcoholism, however not in totality. These animal models include inbred preferring (iP) and nonpreferring (iNP) rat types. Congenic rats have been engineered from the iP and iNP strains whereby a P congenic rat has in its genome a well-chosen chromosomal portion taken from an NP rat (P.NP) and, reciprocally, an NP congenic rat has acquired the analogous DNA from a P rat (NP.P). In this case, a quantitative trait locus (QTL) from chromosome 4 is the donor genetic material for the congenic rats. It is of great interest to further study this chromosome 4 QTL because it has been found to control a significant portion of ethanol consumption behavior in iP and iNP rats. This study aimed to behaviorally profile the iP, iNP and reciprocal congenic rats. As a result of the behavioral profiling of these genetically related groups, some conclusions could be made regarding which behaviors appear to be controlled by the chromosome 4 donor DNA.This study primarily utilized the Multivariate Concentric Square Field apparatus (MCSF) to characterize behavioral profiles for the inbred and congenic rats. The Open field (OF) and Elevated plus maze (EPM) supported this effort. The MCSF is valuable in that it allows for the animals to interact within an environment that has ethological value. The 12 different zones that make up the field are characterized by some functional quality in terms of type and duration of behavior performed, etc. The behavioral data is aggregated and finally represented in terms of five functional categories, the elements of the behavioral profile: general activity, exploratory activity, risk assessment, risk taking, and shelter seeking. The study hypotheses were shaped by prior research suggesting that iPs should display lower general activity and risk taking strategy than iNPs in the MCSF. Inbred Ps should be more active in the OF and spend more time in the center of the EPM. Generally, it is expected that the iP QTL confer behavioral phenotypes to the iNP strain that deviate toward a "P" behavioral phenotype and reciprocally, the iNP QTL confer behavioral phenotypes to the iP strain that deviate toward an "NP" behavioral phenotype. The results showed that iP rats performed more risk assessment and risk taking behavior and less shelter seeking and anxiety-like behavior than iNP rats. It followed that P.NP congenic rats significantly downgraded their risk assessment and risk taking behavior when compared to iP rats. This decrease can be attributed to the chromosome 4 QTL donated from the iNP breed. All together this study concludes that risk assessment and risk taking behavior in the iP rats is controlled by the same DNA region that, in part, determines voluntary intake of ethanol consumption. Further fine mapping of the QTL region should help in discovering if the same DNA sequences that influence ethanol intake also significantly influence risk behavior.Item Characterization of Hepatitis C Virus Infection of Hepatocytes and Astrocytes(2014) Liu, Ziqing; Yu, Andy; He, Johnny J.; Brutkiewicz, Randy R.; Kao, Cheng C.; Sullivan, William J., Jr.Approximately 2.8% of the world population is currently infected with hepatitis C virus (HCV). Neutralizing antibodies (nAbs) are often generated in chronic hepatitis C patients yet fail to control the infection. In the first two chapters of this study, we focused on two alternative routes of HCV transmission, which may contribute to HCV’s immune evasion and establishment of chronic infection. HCV was transmitted via a cell-cell contact-mediated (CCCM) route and in the form of exosomes. Formation of HCV infection foci resulted from CCCM HCV transfer and was cell density-dependent. Moreover, CCCM HCV transfer occurred rapidly, involved all four known HCV receptors and intact actin cytoskeleton, and led to productive HCV infection. Furthermore, live cell imaging revealed the temporal and spatial details of the transfer process. Lastly, HCV from HCV-infected hepatocytes and patient plasma occurred in both exosome-free and exosome-associated forms and the exosome-associated HCV remained infectious, even though HCV infection did not significantly alter exosome secretion. In the third chapter, we characterized HCV interaction with astrocytes, one of the putative HCV target cells in the brain. HCV infection causes the central nervous system (CNS) abnormalities in more than 50% of chronically infected subjects but the underlying mechanisms are largely unknown. We showed that primary human astrocytes (PHA) were very inefficiently infected by HCV, either in the free virus form or through cell-cell contact. PHA expressed all known HCV receptors but failed to support HCV entry. HCV IRES-mediated translation was functional in PHA and further enhanced by miR122 expression. Nevertheless, PHA did not support HCV replication regardless of miR122 expression. To our great surprise, HCV exposure induced robust IL-18 expression in PHA and exhibited direct neurotoxicity. In summary, we showed that CCCM HCV transfer and exosome-mediated HCV infection constituted important routes for HCV infection and dissemination and that astrocytes did not support productive HCV infection and replication, but HCV interactions with astrocytes and neurons alone might be sufficient to cause CNS dysfunction. These findings provide new insights into HCV infection of hepatocytes and astrocytes and shall aid in the development of new and effective strategies for preventing and treating HCV infection.Item Development of a primate model to evaluate the effects of ketamine and surgical stress on the neonatal brain(Sage, 2023) Wang, Cheng; Bhutta, Adnan; Zhang, Xuan; Liu, Fang; Liu, Shuliang; Latham, Leah E.; Talpos, John C.; Patterson, Tucker A.; Slikker, William, Jr.; Pediatrics, School of MedicineWith advances in pediatric and obstetric surgery, pediatric patients are subject to complex procedures under general anesthesia. The effects of anesthetic exposure on the developing brain may be confounded by several factors including pre-existing disorders and surgery-induced stress. Ketamine, a noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist, is routinely used as a pediatric general anesthetic. However, controversy remains about whether ketamine exposure may be neuroprotective or induce neuronal degeneration in the developing brain. Here, we report the effects of ketamine exposure on the neonatal nonhuman primate brain under surgical stress. Eight neonatal rhesus monkeys (postnatal days 5-7) were randomly assigned to each of two groups: Group A (n = 4) received 2 mg/kg ketamine via intravenous bolus prior to surgery and a 0.5 mg/kg/h ketamine infusion during surgery in the presence of a standardized pediatric anesthetic regimen; Group B (n = 4) received volumes of normal saline equivalent to those of ketamine given to Group A animals prior to and during surgery, also in the presence of a standardized pediatric anesthetic regimen. Under anesthesia, the surgery consisted of a thoracotomy followed by closing the pleural space and tissue in layers using standard surgical techniques. Vital signs were monitored to be within normal ranges throughout anesthesia. Elevated levels of cytokines interleukin (IL)-8, IL-15, monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein (MIP)-1β at 6 and 24 h after surgery were detected in ketamine-exposed animals. Fluoro-Jade C staining revealed significantly higher neuronal degeneration in the frontal cortex of ketamine-exposed animals, compared with control animals. Intravenous ketamine administration prior to and throughout surgery in a clinically relevant neonatal primate model appears to elevate cytokine levels and increase neuronal degeneration. Consistent with previous data on the effects of ketamine on the developing brain, the results from the current randomized controlled study in neonatal monkeys undergoing simulated surgery show that ketamine does not provide neuroprotective or anti-inflammatory effects.Item Drinking Rhythms in Alcohol Preferring Mice(2012-08-29) Matson, Liana M.; Grahame, Nicholas J.; Czachowski, Cristine; Boehm II, Stephen L.Multiple lines of High Alcohol Preferring (HAP) mice were selectively bred for their intake of 10% ethanol (v/v) during 24-h daily access over a four-week period, with the highest drinking lines exhibiting intakes in excess of 20 g/kg/day. Drinking rhythms and corresponding blood ethanol concentrations (BEC) of the highest drinking HAP lines to those of the C57BL/6J (B6) inbred strain. Adult male and female crossed HAP (cHAP), HAP1 and B6 mice had free-choice access to 10% ethanol and water for 3 weeks prior to bi-hourly assessments of intake throughout the dark portion of a reverse 12:12 light dark cycle. In another cohort of cHAP mice, the same procedure was used to assess bi-hourly ethanol intake, and blood samples were taken across the day to look at the pattern of accumulation in these mice. Finally, considering the high level of intake by cHAP mice, we were interested in assessing whether metabolic and functional tolerance develop following chronic free-choice access, which were assessed using 2.0 and 1.75 g/kg challenge doses of 20% ethanol, respectively. cHAP and HAP1 mice maintained an excessive level of intake throughout the dark portion of the cycle, accumulating mean BEC levels of 261.5 + 18.09 and 217.9 + 25.02 mg/dl at 7-8 hours following lights off, respectively. B6 mice drank comparatively modestly, and did not accumulate high BEC levels (53.63 + 8.15 mg/dl). In the cHAP cohort, mean BECs were 112.47 + 19.91 at 2 hours after lights off, 189.00 + 27.40 at 6 hours after lights off, 193.80 + 29.66 at 10 hours after lights off, and 89.68 + 22.19 at 2 hours after lights on. Further, following 3 weeks of ethanol access, cHAP mice had a faster rate of ethanol metabolism and fewer hind slips than water-only exposed mice (ps < .05). In conclusion, the excessive free-choice drinking demonstrated by the HAP1 and cHAP lines, as well as the pattern of sustained high BECs in cHAP mice, challenge the notion that rodents will not reliably and voluntarily sustain ethanol intake at pharmacologically relevant levels. These results suggest that the highest drinking HAP lines may provide a unique opportunity for modeling the excessive intake that has been observed in alcohol-dependent individuals. Further, we observed that cHAP mice develop both metabolic and functional tolerance to the ataxic effects of ethanol following 3 weeks of free-choice access. Together, these findings support HAP mice as translational rodent model of alcoholism, and provide rationale for exploration of the predisposing factors for excessive consumption, as well as the development of physiological, behavioral, and toxicological outcomes following alcohol exposure.Item Dual regulation of voltage- and ligand-gated calcium channels by collapsin response mediator protein 2(2013-10-07) Brittain, Joel Matthew; Khanna, Rajesh; Cummins, Theodore R.; Oxford, G. S.; Quilliam, Lawrence; Thurmond, Debbie C.Synaptic transmission is coordinated by a litany of protein-protein interactions that rely on the proper localization and function of pre- and post-synaptic Ca2+ channels. The axonal guidance/specification collapsin response mediator protein-2 (CRMP-2) was identified as a potential partner of the pre-synaptic N-type voltage-gated Ca2+ channel (CaV2.2). CRMP-2 bound directly to CaV2.2 in two regions; the channel domain I-II intracellular loop and the distal C-terminus. Both proteins co-localized within presynaptic sites in hippocampal neurons. Overexpression in hippocampal neurons of a CRMP-2 protein fused to EGFP caused a significant increase in Ca2+ channel current density whereas lentivirus-mediated CRMP-2 knockdown abolished this effect. Cell surface biotinylation studies showed an increased number of CaV2.2 at the cell surface in CRMP-2–overexpressing neurons. Both activity- and CRMP-2-phosphoryation altered the interaction between CaV2.2 and CRMP-2. I identified a CRMP-2-derived peptide (called CBD3) that bound CaV2.2 and effectively disrupted the interaction between CaV2.2 and CRMP-2. CBD3 peptide fused to the HIV TAT protein (TAT-CBD3) decreased neuropeptide release from sensory neurons and excitatory synaptic transmission in dorsal horn neurons, and reversed neuropathic hypersensitivity produced by an antiretroviral drug. Unchecked Ca2+ influx via N-methyl-D-aspartate receptors (NMDARs) has been linked to activation of neurotoxic cascades culminating in cell death (i.e. excitotoxicity). CRMP-2 was suggested to affect NMDAR trafficking and possibly involved in neuronal survival following excitotoxicity. Based upon these studies, I hypothesized that a peptide from CRMP2 could preserve neurons in the face of excitotoxic challenges. Lentiviral–mediated CRMP2 knockdown or treatment with TAT-CBD3 blocked neuronal death following glutamate exposure likely via blunting toxicity from NMDAR-mediated delayed calcium deregulation. TAT-CBD3 induced internalization of the NMDAR subunit NR2B in dendritic spines without altering somal surface expression. TAT-CBD3 reduced NMDA-mediated Ca2+-influx and currents in cultured neurons. The presented work validates CRMP-2 as a novel modulator of pre- and post-synaptic Ca2+ channels and provides evidence that the TAT-CBD3 peptide could be useful as a potential therapeutic for both chronic neuropathic pain and excitotoxicity following stroke or other neuronal insults.Item The effects of CaMKII signaling on neuronal viability(2013-12-10) Ashpole, Nicole M.; Hudmon, Andrew; Brustovetsky, Nickolay; Hurley, Thomas D., 1961-; Russell, Weihua Lee, 1956-; Oxford, G. S.Calcium/calmodulin-dependent protein kinase II (CaMKII) is a critical modulator of synaptic function, plasticity, and learning and memory. In neurons and astrocytes, CaMKII regulates cellular excitability, cytoskeletal structure, and cell metabolism. A rapid increase in CaMKII activity is observed within the first few minutes of ischemic stroke in vivo; this calcium-dependent process is also observed following glutamate stimulation in vitro. Activation of CaMKII during pathological conditions is immediately followed by inactivation and aggregation of the kinase. The extent of CaMKII inactivation is directly correlated with the extent of neuronal damage. The studies presented here show that these fluctuations in CaMKII activity are not correlated with neuronal death; rather, they play a causal role in neuronal death. Pharmacological inhibition of CaMKII in the time immediately surrounding glutamate insult protects cultured cortical neurons from excitotoxicity. Interestingly, pharmacological inhibition of CaMKII during excitotoxic insult also prevents the aggregation and prolonged inactivation of the kinase, suggesting that CaMKII activity during excitotoxic glutamate signaling is detrimental to neuronal viability because it leads to a prolonged loss of CaMKII activity, culminating in neuronal death. In support of this, CaMKII inhibition in the absence of excitotoxic insult induces cortical neuron apoptosis by dysregulating intracellular calcium homeostasis and increasing excitatory glutamate signaling. Blockade of the NMDA-receptors and enzymatic degradation of the extracellular glutamate signal affords neuroprotection from CaMKII inhibition-induced toxicity. Co-cultures of neurons and glutamate-buffering astrocytes also exhibit this slow-induced excitotoxicity, as CaMKII inhibitors reduce glutamate uptake within the astrocytes. CaMKII inhibition also dysregulates calcium homeostasis in astrocytes and leads to increased ATP release, which was neurotoxic when applied to naïve cortical neurons. Together, these findings indicate that during aberrant calcium signaling, the activation of CaMKII is toxic because it supports aggregation and prolonged inactivation of the kinase. Without CaMKII activity, neurons and astrocytes release stores of transmitters that further exacerbate neuronal toxicity.Item Effects of Developmental Low-Level Lead Exposure on Voluntary Alcohol Consumption and Drug-Induced Behavioral Sensitization in Adulthood(2020-12) Hernández, Maribel; Boehm, Stephen; Filippelli, Gabriel; Graham, NicholasLead (Pb) is one of the most harmful and most abundant neurotoxins in the environment. Despite the extensive movement made to eradicate toxic levels of Pb in the environment, children, predominately in lower socioeconomic areas, are still exposed to varying levels of Pb. Human studies suggest that Pb exposure leads to altered drug consumption in adults by altering underlying neural mechanisms, specifically dopamine (DA) activity. However, there is limited research on this at blood Pb levels below 10 μg/dL, levels often seen in children growing up in neighborhoods located in old industrial and urban areas. To model how early-life low-level Pb exposure effects DA-dependent behaviors associated with addiction in adulthood, we used C57BL/6J mice. Litters were weaned at PND 21 and assigned to either a three-week history of 30 parts per million (ppm) Lead (IV) Acetate exposure or a control condition of 0 ppm Pb in DI drinking water. After the Pb exposure period, mice were switched to regular tap water until they reached adulthood. Afterward, separate animals were tested in one of three experiments: two-bottle choice alcohol preference drinking, alcohol-induced behavioral sensitization (EBS), and cocaine-induced behavioral sensitization (CBS). In experiment 1, our hypothesis was met, and both male and female mice with a prior Pb exposure displayed significantly higher alcohol intake and preference scores over the three-week period than control mice. In experiment 2, there were no differences in EBS and no evidence of EBS in any of the groups. However, there was an increased acute response to 2.0 g/kg EtOH in the Pb-exposed chronic group as compared to the control animals. Lastly, in experiment 3, Pb-exposed animals in the chronic cocaine group were more sensitive to the effects of cocaine (10 mg/kg) across days than the controls, both the acute cocaine groups and both saline control groups. Thus, with these experiments, we concluded that low levels of developmental Pb exposure might be targeting DA in the reward pathway, which is essential for alcohol intake and drug sensitization.Item Peri-adolescent Alcohol Consumption Enhances the Reinforcing and Stimulatory Properties of Ethanol within the Adult Mesolimbic Dopamine System in Alcohol Preferring P Rats(2012-08-07) Toalston, Jamie E.; Murphy, James M.; Neal-Beliveau, Bethany S.; Bell, Richard L.; Kinzig, Kimberly P.Research in the alcohol preferring (P) rat has indicated that peri-adolescent alcohol (EtOH) consumption enhances the acquisition of oral operant EtOH self-administration, inhibits the extinction of responding for EtOH, augments EtOH-seeking behaviors, and increases relative reward value of EtOH during adulthood. Experiment 1 was conducted to determine if these adult effects of peri-adolescent EtOH intake could be observed using an Intracranial Self-Administration (ICSA) model. It was hypothesized that an increased sensitivity to the rewarding actions of EtOH would be manifested in peri-adolescent-EtOH-exposed subjects compared to naive subjects when the opportunity to self-administer EtOH to the posterior ventral tegmental area (pVTA) is available in adulthood. The pVTA is a primary site for EtOH’s reinforcing and rewarding properties in the mesolimbic dopamine (DA) system. Experiment 2 was a dose-response examination of the effects of EtOH administered to the pVTA on downstream DA efflux in the nucleus accumbens shell (AcbSh) via a joint Microinjection-Microdialysis (MicroMicro) procedure. Male P rats were given 24-h free-choice exposure to 15% volume/volume EtOH from postnatal day (PD) 30 to PD 60, or remained experimentally naive, with ad lib food and water. By the end of the periadolescent exposure period, average consumption was 7.3 g/kg/day of EtOH. After PD 75, periadolescent-EtOH-exposed and naïve rats were either implanted with an injector guide cannula aimed at the right pVTA for ICSA (Experiment 1), or two cannulae, one aimed at the right pVTA (injector) and one at the ipsilateral AcbSh (microdialysis) for MicroMicro (Experiment 2). Following one week of recovery from surgery, ICSA subjects were placed in standard two-lever (active and inactive) operant chambers. Test sessions were 60 min in duration and occurred every other day for a total of 7 sessions. Rats were randomly assigned to one of 5 groups (n=4-9/group) that self-infused (FR1 schedule) either aCSF (vehicle, 0 mg%), 50, 75, 100, or 150 mg% EtOH during 4 sessions, aCSF only for sessions 5 and 6 (extinction), and the initial concentration again for session 7 (reinstatement). MicroMicro subjects received six days of recovery from surgery, probe implantation the day before testing, and then continuous microdialysis for DA with 15 min microdialysis samples collected before, during, and then two hrs after 10-min pulse microinjection of either aCSF (vehicle, 0 mg%), 50, 75, 100, or 150 mg% EtOH. Neither EtOH-exposed nor naive groups of P rats self-infused the aCSF or 50 mg% EtOH concentration. While the naive group did not self-infuse the 75 or 100 mg% EtOH concentrations, the peri-adolescent EtOH-exposed group of P rats did readily discriminate the active lever from the inactive lever at these concentrations. Both groups self-infused the 150 mg% EtOH concentration. Pulse microinjections of EtOH during the MicroMicro procedure revealed that 75 and 100 mg% concentrations of EtOH increased downstream DA in the AcbSh of EtOH-exposed, but not naïve, subjects. 150 mg% EtOH increased downstream DA in both adolescent treatment groups. Overall, the results indicate that consumption of EtOH by P rats during peri-adolescence increases the reinforcing properties of EtOH in the pVTA in adulthood. The results also indicate that there were differential effects of peri-adolescent EtOH exposure on DA efflux in the AcbSh. This provides evidence that peri-adolescent EtOH-exposure produces long-lasting alterations in neural circuitry involved in EtOH-reinforcement, during adulthood.