Medial Prefrontal Cortical Dopamine Responses During Operant Self-Administration of Sweetened Ethanol.

BACKGROUND: Medial prefrontal cortex (mPFC) dysfunction is present in heavy alcohol consumers. Dopamine signaling in mPFC is associated with executive functioning and affects drinking behavior; however, direct measurement of extracellular mPFC dopamine during appetitive and consummatory ethanol (EtOH) self-administration behavior has not been reported. METHODS: We used in vivo microdialysis in freely behaving, adult, male, Long Evans rats to determine extracellular dopamine concentration in the mPFC during operant self-administration of an EtOH-plus-sucrose or sucrose solution. The model separated appetitive/seeking from consummatory phases of the operant session. Dopamine was also monitored in an untrained handling control group, and dialysate EtOH was measured in the EtOH-drinking group. RESULTS: Home cage baseline dopamine was lower in rats that experienced a week of drinking sweetened EtOH compared with sucrose-drinking and handling controls. Transfer into the operant chamber and the initiation of consumption stimulated a relatively higher change in dopamine over baseline in the sweetened EtOH group compared with sucrose and handling controls. However, all groups show a dopamine response during transfer into the operant chamber, and the sucrose group had a relatively higher change in dopamine over baseline during initiation of consumption compared with handling controls. The time courses of dopamine and EtOH in the mPFC differ in the EtOH-consuming rats. CONCLUSIONS: Differences in extracellular mPFC dopamine between EtOH drinkers compared with control groups suggest that mPFC dopamine is involved in the mechanism of operant self-administration of sweetened EtOH and sucrose. Furthermore, the increase in dopamine during consumption is consistent with a role of mPFC dopamine in reward prediction.

Operant self-administration of sweetened ethanol and time course of blood ethanol levels in adolescent and adult male Long-Evans rats.

BACKGROUND: Little is known regarding mechanisms regulating ethanol (EtOH) self-administration during adolescence or if the mechanisms differ from adults. One of the best models of abuse liability is operant self-administration. Therefore, we characterized operant sweetened EtOH self-administration behavior in adolescent and adult rats. METHODS: Adolescent (36 days old at first EtOH exposure) and adult male Long-Evans rats were first trained to self-administer 10% sucrose (10S) in an appetitive/consummatory operant model for 1 week, and then the drinking solution was switched to 10% sucrose plus 10% EtOH (10S10E) for 2 weeks. Next, rats were switched to a fixed ratio 2 schedule, and this was followed by 1 session using a progressive ratio schedule of reinforcement. Last, rats were tested for cue-induced reinstatement of lever-pressing behavior under extinction conditions after 13 days of abstinence. Blood EtOH concentration (BEC) of sweetened EtOH (self-administered or intragastric [IG] administration of 1 g/kg) was determined via gas chromatography. Control rats drank only 10S. RESULTS: Consumption of sweetened EtOH was not different between adolescents and adults under any schedule tested, reaching 1 g/kg in 20 minutes in the appetitive/consummatory model. Appetitive behavior directed at sweetened EtOH was less focused in adolescents versus adults. No age differences were found in motivation for sweetened EtOH. Cue-induced reinstatement of EtOH-seeking behavior after abstinence also did not differ by age. In control groups, no age difference was found in appetitive behavior or the amount of sucrose consumed, although adults exhibited greater cue-induced reinstatement. BEC after self-administration or IG administration of sweetened EtOH was higher in adults than adolescents. CONCLUSIONS: Consumption and motivation for sweetened EtOH are similar in adolescents and adults, although adolescents are more vulnerable to the effects of EtOH consumption on appetitive behavior. The IG results suggest larger volume of distribution and higher first-pass metabolism of sweetened EtOH in adolescents versus adults, which may limit the reinforcing effects of EtOH in some adolescents. Overall, we have begun to establish an operant sweetened EtOH self-administration model in adolescent rats.

Diversity-function relationships changed in a long-term restoration experiment.

The central tenet of biodiversity-ecosystem function (BEF) theory, that species richness increases function, could motivate restoration practitioners to incorporate a greater number of species into their projects. But it is not yet clear how well BEF theory predicts outcomes of restoration, because it has been developed through tests involving short-run and tightly controlled (e.g., weeded) experiments. Thus, we resampled our 1997 BEF experiment in a restored salt marsh to test for long-term effects of species richness (plantings with 1, 3, and 6 species per 2 x 2 m plot), with multiple ecosystem functions as response variables. Over 11 years, 1- and 6-species assemblages converged on intermediate richness (mean = 3.9 species/ 0.25-m2 plot), and composition changed nonrandomly throughout the site. While three species became rare, the two most productive species became co-dominant. The two dominants controlled and increased shoot biomass, which appeared to decrease species richness. Diversity-function relationships became less positive over 11 years and differed significantly with (a) the species-richness metric (planted vs. measured), and (b) the indicator of function (shoot biomass, height, and canopy layering). The loss of positive relationships between species richness and function in our restored site began soon after we stopped weeding and continued with increasing dominance by productive species. Where species-rich plantings are unlikely to ensure long-term restoration of functions, as in our salt marsh, we recommend dual efforts to establish (1) dominant species that provide high levels of target functions, and (2) subordinate species, which might provide additional functions under current or future conditions.

Correction to: Acute phenylalanine/tyrosine depletion of phasic dopamine in the rat brain.

After publication of this paper, the authors determined an error in the calculation of the norepinephrine standard concentrations for the HPLC calibration curves.

High sensitivity HPLC method for analysis of in vivo extracellular GABA using optimized fluorescence parameters for o-phthalaldehyde (OPA)/sulfite derivatives.

Reversed-phase HPLC with derivatization using o-phthalaldehyde (OPA) and sulfite allows electrochemical detection of γ-aminobutyric acid (GABA) in microdialysis samples. However, OPA/sulfite derivatives have been reported to produce lower fluorescent yield than OPA derivatives using organic thiols as the nucleophile. To overcome this limitation we examined excitation and emission spectra, reaction time, pH, and concentration of reagents in the derivatization solution. Optimal detection parameters were determined as λex=220nm and λem=385nm for maximal fluorescence. The derivatization reaction occurred immediately and the product was stable up to 24 h [corrected]. A pH of 10.4 for the borate buffer used in the derivatization solution was significantly better than lower pH. Increasing the amount of sulfite combined with diluting the derivatization solution in borate buffer resulted in complete separation of the GABA peak from contaminants without any loss in signal. Controlling the temperature of the detector at 15°C significantly improved sensitivity with a detection limit of approximately 1nM. To validate this assay, we performed microdialysis in the dorsal striatum and ventral tegmental area (VTA) of adult Long Evans rats. GABA concentrations in dialysates were determined using external standards and standard additions, in order to further confirm interfering peaks were not present in biological samples. Within the dorsal striatum (n=4), basal GABA concentrations were 12.9±2.2 and 14.5±2.2nM (external and additions, respectively). Respective basal GABA concentrations in the VTA (n=3) were 4.6±1.1 and 5.1±0.6nM. Thus, we have developed a novel, sensitive fluorescence method to determine GABA in microdialysates using HPLC of an OPA/sulfite derivative.

Acute phenylalanine/tyrosine depletion of phasic dopamine in the rat brain.

RATIONALE: Dopamine plays a critical role in striatal and cortical function, and depletion of the dopamine precursors phenylalanine and tyrosine is used in humans to temporarily reduce dopamine and probe the role of dopamine in behavior. This method has been shown to alter addiction-related behaviors and cognitive functioning presumably by reducing dopamine transmission, but it is unclear what specific aspects of dopamine transmission are altered. OBJECTIVES: We performed this study to confirm that administration of an amino acid mixture omitting phenylalanine and tyrosine (Phe/Tyr[-]) reduces tyrosine tissue content in the prefrontal cortex (PFC) and nucleus accumbens (NAc), and to test the hypothesis that Phe/Tyr[-] administration reduces phasic dopamine release in the NAc. METHODS: Rats were injected with a Phe/Tyr[-] amino acid mixture, a control amino acid mixture, or saline. High-performance liquid chromatography was used to determine the concentration of tyrosine, dopamine, or norepinephrine in tissue punches from the PFC and ventral striatum. In a separate group of rats, phasic dopamine release was measured with fast-scan cyclic voltammetry in the NAc core after injection with either the Phe/Tyr[-] mixture or the control amino acid solution. RESULTS: Phe/Tyr[-] reduced tyrosine content in the PFC and NAc, but dopamine and norepinephrine tissue content were not reduced. Moreover, Phe/Tyr[-] decreased the frequency of dopamine transients, but not their amplitude, in freely moving rats. CONCLUSIONS: These results indicate that depletion of tyrosine via Phe/Tyr[-] decreases phasic dopamine transmission, providing insight into the mechanism by which this method modifies dopamine-dependent behaviors in human imaging studies.

Lack of effect of nucleus accumbens dopamine D1 receptor blockade on consumption during the first two days of operant self-administration of sweetened ethanol in adult Long-Evans rats.

The mechanisms underlying ethanol self-administration are not fully understood; however, it is clear that ethanol self-administration stimulates nucleus accumbens dopamine release in well-trained animals. During operant sweetened ethanol self-administration behavior, an adaptation in the nucleus accumbens dopamine system occurs between the first and second exposure, paralleling a dramatic increase in sweetened ethanol intake, which suggests a single exposure to sweetened ethanol may be sufficient to learn the association between sweetened ethanol cues and its reinforcing properties. In the present experiment, we test the effects of blockade of nucleus accumbens dopamine D1 receptors on operant sweetened ethanol self-administration behavior during the first 2 days of exposure. Adult male Long-Evans rats were first trained to self-administer 10% sucrose (10S) across 6 days in an appetitive and consummatory operant model (appetitive interval: 10-min pre-drinking wait period and a lever response requirement of 4; consummatory interval: 20-min access to the drinking solution). After training on 10S, the drinking solution was switched to 10% sucrose plus 10% ethanol (10S10E); control rats continued drinking 10S throughout the experiment. Bilateral nucleus accumbens microinjections of the dopamine D1 antagonist, SCH-23390 (0, 1.0, or 3.0 µg/side), immediately preceded the first two sessions of drinking 10S10E. Results show that blocking nucleus accumbens dopamine D1 receptors has little or no influence on consumption during the first 2 days of exposure to the sweetened ethanol solution or maintenance of sucrose-only drinking. Furthermore, the high dose of SCH-23390, 3.0 µg/side, reduced open-field locomotor activity. In conclusion, we found no evidence to suggest that nucleus accumbens D1 receptor activation is involved in consumption of a sweetened ethanol solution during the first 2 days of exposure or maintenance of sucrose drinking, but rather D1 receptors seem necessary for general locomotor activity that contributes to initiation of appetitive behavior.

Attenuated effects of experimenter-administered heroin in adolescent vs. adult male rats: physical withdrawal and locomotor sensitization.

OBJECTIVES: Early onset of heroin use during adolescence might increase chances of later drug addiction. Prior work from our laboratory suggests, however, that adolescent male rats are actually less sensitive than adults to some enduring effects of heroin self-administration. In the present study, we tested two likely correlates of sensitivity to behavioral reinforcement in rats: physical withdrawal and locomotor sensitization. METHODS: Adolescent (35 days old at start) and adult (79 days old) male Sprague-Dawley rats were administered escalating doses of heroin, increasing from 1.0 to 8.0 mg/kg (i.p.) every 12 h, across 13 days. Somatic signs of spontaneous withdrawal were scored 12 and 24 h after the last injection, and then every 24 h for 5 days; locomotion was recorded concurrently. Challenge injections of heroin (1 mg/kg i.p.) were given at four points: as the first of the escalating doses (day 1), at days 7 and 13 during the escalating regimen, and after 12 days of forced abstinence. Body mass and food intake were measured throughout experimentation. RESULTS: A heroin withdrawal syndrome was not observed among adolescents as it was among adults, including somatic signs as well as reduced locomotion, body mass, and food intake. On the other hand, heroin-induced locomotor sensitization did not differ across ages. CONCLUSION: Reduced withdrawal is consistent with the attenuated reinforcing effects of heroin among adolescent male rats that we reported previously. Thus, it is possible that adolescent rats could reveal important neuroprotective factors for use in treatment of heroin dependence.

A role for the prefrontal cortex in heroin-seeking after forced abstinence by adult male rats but not adolescents.

Adolescent drug abuse is hypothesized to increase the risk of drug addiction. Yet male rats that self-administer heroin as adolescents show attenuated drug-seeking after abstinence, compared with adults. Here we explore a role for neural activity in the medial prefrontal cortex (mPFC) in age-dependent heroin-seeking. Adolescent (35-day-old at start; adolescent-onset) and adult (86-day-old at start) male rats acquired lever-pressing maintained by heroin using a fixed ratio one reinforcement schedule (0.05 and 0.025 mg/kg per infusion). Following 12 days of forced abstinence, rats were tested for heroin-seeking over 1 h by measuring the number of lever presses on the active lever. Unbiased stereology was then used to estimate the number of Fos-ir(+) and Fos-ir(-) neurons in prelimbic and infralimbic mPFC. As before, adolescents and adults self-administered similar amounts of heroin, but subsequent heroin-seeking was attenuated in the younger rats. Similarly, the adolescent-onset group failed to show significant neural activation in the prelimbic or infralimbic mPFC during the heroin-seeking test, whereas the adult-onset heroin self-administration group showed two to six times more Fos-ir(+) neurons than their saline counterparts in both mPFC subregions. Finally, the overall number of neurons in the infralimbic cortex was greater in rats from the adolescent-onset groups than adults. The mPFC may thus have a key role in some age-dependent effects of heroin self-administration.

Heroin self-administration and reinstatement of heroin-seeking in adolescent vs. adult male rats.

RATIONALE: Heroin abuse is prevalent among teenagers, and early onset drug use might predict long-term drug dependence. However, adolescent sensitivity to drug reinforcement has not been explored thoroughly in animal models. OBJECTIVES: This study aimed to compare intravenous (i.v.) self-administration of heroin, as well as extinction and reinstatement of heroin-seeking, in adolescent vs. adult male rats. METHODS: Adolescent (35 days old at start) and adult (86 days old at start) male Sprague-Dawley rats spontaneously acquired lever pressing maintained by i.v. heroin infusions. In experiment 1, self-administration was tested on a fixed ratio 1 schedule of reinforcement (0.05 and 0.025 mg/kg per infusion), followed by within-session extinction and reinstatement tests after 1 or 12 days of abstinence. In experiment 2, self-administration was tested on a progressive ratio schedule (0.0125-0.1 mg/kg per infusion), followed 12 days later by a single test of extinction responding in the presence of cues. RESULTS: In experiment 1, adolescent rats self-administered more heroin than adults. After 1 or 12 days of abstinence, adolescents exhibited less heroin-seeking than adults, although levels of heroin-seeking increased over abstinence period for both age groups. In experiment 2, adolescents and adults reached the same maximal response ratio (breakpoint), although adolescents earned more infusions when response requirements were low. For extinction responding in the presence of cues, heroin-seeking was similar across ages. CONCLUSIONS: Lower levels of heroin-seeking suggest that younger rats are less sensitive than adults to some residual effects of heroin intake.

A high fructose diet does not affect amphetamine self-administration or spatial water maze learning and memory in female rats.

High energy diets can have a detrimental effect on brain plasticity. For example, a high fructose diet impairs spatial memory in male rats. The aim of the present study was to determine whether a high fructose diet impairs another form of learning and memory: drug reinforcement learning. Female Sprague-Dawley rats were fed a high fructose diet (60%) from weaning at postnatal day (PND) 21, then allowed to acquire lever-pressing maintained by intravenous (i.v.) amphetamine at PND 68, 109, or 165. Acquisition was tested on a fixed ratio one (FR1) schedule of reinforcement (0.025 mg/kg/infusion, 1h daily sessions, 10 sessions over 14 days), followed by testing for reinforcing efficacy on a progressive ratio (PR) schedule (0.025, 0.01, and 0.1mg/kg/infusion), 14 days of abstinence, and within-session extinction and reinstatement tests. Subsequently, water maze acquisition and retention were tested in these subjects as well as a separate cohort tested in the water maze only. The diet had no effect on acquisition, reinforcing efficacy, extinction, or reinstatement of amphetamine seeking. Nor did the diet alter any measures of spatial memory. The high fructose diet did decrease body mass and increase relative liver and spleen mass, but did not affect plasma triglyceride concentrations consistently. Together with prior research on males, these results suggest that the metabolism of fructose and the effects of a high fructose diet on learning and memory may be sex-dependent.

Contributions of dopamine D1, D2, and D3 receptor subtypes to the disruptive effects of cocaine on prepulse inhibition in mice.

Deficits in prepulse inhibition (PPI) of startle, an operational measure of sensorimotor gating, are characteristics of schizophrenia and related neuropsychiatric disorders. Previous studies in mice demonstrate a contribution of dopamine (DA) D(1)-family receptors in modulating PPI and DA D(2) receptors (D2R) in mediating the PPI-disruptive effects of amphetamine. To examine further the contributions of DA receptor subtypes in PPI, we used a combined pharmacological and genetic approach. In congenic C57BL/6 J wild-type mice, we tested whether the D1R antagonist SCH23390 or the D2/3R antagonist raclopride would attenuate the effects of the indirect DA agonist cocaine (40 mg/kg). Both the D1R and D2/3R antagonists attenuated the cocaine-induced PPI deficit. We also tested the effect of cocaine on PPI in wild-type and DA D1R, D2R, or D3R knockout mice. The cocaine-induced PPI deficit was influenced differently by the three DA receptor subtypes, being absent in D1R knockout mice, partially attenuated in D2R knockout mice, and exaggerated in D3R knockout mice. Thus, the D1R is necessary for the PPI-disruptive effects of cocaine, while the D2R partially contributes to these effects. Conversely, the D3R appears to inhibit the PPI-disruptive effects of cocaine. Uncovering neural mechanisms involved in PPI will further our understanding of substrates of sensorimotor gating and could lead to better therapeutics to treat complex cognitive disorders such as schizophrenia.

Antagonistic effects of dopaminergic signaling and ethanol on protein kinase A-mediated phosphorylation of DARPP-32 and the NR1 subunit of the NMDA receptor.

BACKGROUND: This article extends our initial investigation of the interactions between dopamine and glutamate receptor systems after acute exposure to ethanol. DARPP-32 (dopamine and cyclic adenosine monophosphate-regulated phosphoprotein of approximate molecular weight 32 kDa) is an important regulator of protein phosphatase-1 that in turn regulates a large number of effectors, including the NMDA receptor. METHODS: We measured the protein kinase A (PKA)-mediated phosphorylation of DARPP-32 and the NR1 subunit of the NMDA receptor. Initially, corpus striatum was assayed after intraperitoneal treatment of mice with the D1 agonist SKF82958, the D2 agonist and anticraving drug bromocriptine, or ethanol. In other experiments we blocked D1 receptors with the selective D1 antagonist SCH23390 or blocked D2 receptors with the selective D2 antagonist eticlopride. Finally, we examined combinations of some dopaminergic drugs with and without ethanol. RESULTS: SKF82958 alone significantly increased PKA-mediated phosphorylation of both DARPP-32 and NR1. Bromocriptine alone had no effect on pDARPP-32 or on pNR1. However, when D1 receptors were blocked, bromocriptine reduced the PKA-mediated phosphorylation of both DARPP-32 and NR1. Coincident treatment with bromocriptine and ethanol reversed both of these effects with D1 receptors blocked. The combination of eticlopride (D2 blocker) and SF82958 (D1 agonist) did not significantly alter either pDARPP-32 or pNR1. CONCLUSIONS: These data demonstrate antagonistic effects of acute ethanol exposure on D1 signaling in vivo and the potential of acute in vivo challenge protocols to help fill gaps in the understanding of ethanol's effects on protein phosphorylation.