Increased ethanol excitation of dopaminergic neurons of the ventral tegmental area after chronic ethanol treatment

BACKGROUND: The mesolimbic dopamine pathway, which originates in the ventral tegmental area (VTA), is important for the rewarding effects of ethanol. Acute administration of ethanol has been shown to excite dopaminergic neurons of the VTA. Chronic ethanol treatment has been reported to alter the in vitro response of dopaminergic neurons to NMDA and dopamine. The present electrophysiological study tested the hypothesis that the effect of ethanol, gamma-aminobutyric acid (GABA), and NMDA on individual dopaminergic VTA (DA-VTA) neurons from C57BL/6J mice would be changed by chronic treatment with ethanol. METHODS: C57BL/6J mice were injected intraperitoneally twice daily with either saline or ethanol in saline (3.5 g/kg) for at least 21 days. Extracellular single unit recordings of spontaneous action potentials were made from DA-VTA neurons in brain slices from these mice. Ethanol (20-120 mM), GABA (50-500 microM), or NMDA (2-20 microM) was administered in the superfusate, and the resulting change in firing rate was measured. RESULTS: There was no significant difference in mean basal spontaneous firing rate of DA-VTA neurons between saline-treated and ethanol-treated mice. The DA-VTA neurons from ethanol-treated mice were excited by ethanol more potently than those from saline-treated mice. Dopaminergic VTA neurons from ethanol-treated mice were inhibited less potently by GABA than those from saline-treated mice. There was no difference in the potency of NMDA to excite DA-VTA neurons from saline-treated and ethanol-treated mice. CONCLUSIONS: Chronic treatment of C57BL/6J mice with ethanol injections sensitizes DA-VTA neurons to ethanol excitation and also decreases the inhibitory potency of GABA. The increase in sensitivity to ethanol excitation of dopaminergic VTA neurons after chronic ethanol treatment may increase the reward value of ethanol. This sensitization to ethanol activation may be an important change in reward area neurons and may contribute to the development of alcoholism.     

Enduring effects of chronic ethanol in the CNS: basis for alcoholism

This symposium focused on functional alterations in the mesolimbic dopamine system during the abstinence phase after chronic alcohol intake. Mark Brodie first described his recordings from midbrain slices prepared after chronic alcohol treatment in vivo by daily injection in C57BL/6J mice. No changes were found in the baseline firing frequency of dopaminergic neurones in the VTA (ventral tegmental area), but the excitation produced in these neurones by an acute ethanol challenge was significantly increased in neurons from ethanol-treated mice compared with those from the saline-treated controls. There was also a significant decrease in the inhibitory response to GABA by the dopamine neurones following the chronic ethanol treatment. These data suggest that the timing pattern and mode of ethanol administration may determine the types of changes observed in dopaminergic reward area neurons. Annalisa Muntoni lectured on the relationship between electrophysiological and biochemical in vivo evidence supporting a reduction in tonic activity of dopamine neurons projecting to the nucleus accumbens at various times after suspension of chronic ethanol treatment and morphological changes affecting dopamine neurons in rat VTA. Hilary J. Little then described changes in dopaminergic neurone function in the VTA during the abstinence phase. Decreases in baseline firing were seen at 6 days after withdrawal of mice from chronic ethanol treatment but were not apparent after 2 months abstinence. Increases in the affinity of D1 receptors in the striatum, but not in the cerebral cortex, were seen however up to 2 months after withdrawal. Scott Steffensen then described his studies recording in vivo from GABA containing neurones in the VTA in freely moving rats. Chronic ethanol administration enhanced the baseline activity of these neurones and resulted in tolerance to the inhibition by ethanol of these neurones. His results demonstrated selective adaptive circuit responses within the VTA or in extrategmental structures that regulate VTA-GABA neurone activity.     

Discriminative stimulus effects of ethanol in C57BL/6J and DBA/2J inbred mice

BACKGROUND: Two of the most widely used mouse strains for studying the behavioral effects of ethanol are C57BL/6J (B6) and DBA/2J (D2) mice. These strains exhibit marked differences in behavioral and physiological responses to ethanol. The subjective discriminative stimulus effects of ethanol may play a role in ethanol abuse, but the discriminative stimulus profile of ethanol has not been compared in B6 and D2 mice. Examination of the discriminative stimulus effects of ethanol in B6 and D2 mouse strains may enhance our understanding of the relationship between the subjective effects of ethanol and other ethanol-induced behavioral effects. METHODS: Twelve adult male C57BL/6J mice and 12 male DBA/2J mice were trained to discriminate 1.5 g/kg ethanol from saline in daily 15 min, milk-reinforced operant sessions. After training, ethanol substitution and response-rate suppression dose response curves were determined for ethanol, midazolam, diazepam, pentobarbital, pregnanolone, 4,5,6,7-Tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), dizocilpine, and morphine. RESULTS: D2 mice learned the ethanol discrimination significantly more quickly than did B6 mice. Ethanol, midazolam, pregnanolone, and dizocilpine fully substituted for ethanol in both strains. Pentobarbital was more potent in producing ethanol-like discriminative stimulus effects in D2 than B6 mice. Midazolam and diazepam were significantly more potent in suppressing response rates in D2 than B6 mice. Morphine failed to substitute for ethanol in either strain, but the ED50 for morphine suppression of responding was significantly lower in B6 than D2 mice. CONCLUSIONS: The initial stimulus effects of 1.5 g/kg ethanol may be more salient in D2 than B6 mice. This does not appear to result from differences in the neurotransmitter systems that mediate ethanol's discriminative stimulus effects. In both strains, gamma-aminobutyric acid-positive modulators and a noncompetitive NMDA antagonist substituted for ethanol. However, strain differences did exist in the potency of gamma-aminobutyric acid-positive modulators and morphine for suppressing operant responding.     

Role of genotype in the development of locomotor sensitization to alcohol in adult and adolescent mice: comparison of the DBA/2J and C57BL/6J inbred mouse strains

Background: Animal models that explore differential sensitivity to the effects of acute and repeated exposure of alcohol (ethanol) may be influenced by both the developmental and genetic profile of the population. Therefore, we sought to compare the influence of ontogeny on sensitivity to ethanol‐induced locomotor stimulation and on the induction of locomotor sensitization to this effect across 2 inbred strains of mice; the ethanol consuming C57BL/6J and the ethanol avoiding DBA/2J strains. Methods: C57BL/6J and DBA/2J adults (postnatal day [PD] 60 to 80) and adolescents (PD 30 ± 2) were assessed for basal activity, acute response to 2.0 g/kg ethanol, and the expression of locomotor sensitization following repeated administration of 2.5, 3.0, or 3.5 g/kg ethanol. Results: Basal activity was different across development for the C57BL/6J, but not DBA/2J, with adult B6 mice showing persistently greater baseline activity. Adolescents of both strains were more sensitive than adults to acute ethanol‐induced locomotor stimulation; adults exhibited a decrease in their acute response across the testing session. Adolescent DBA/2J mice developed less ethanol sensitization compared to adults, with significant sensitization observed only following repeated administration of the lowest ethanol dose (2.5 g/kg), whereas DBA/2J adults sensitized to all doses. Age did not influence the development of ethanol sensitization for the C57BL/6J strain, as both adults and adolescents displayed a sensitized response following all ethanol doses. Conclusions: These results suggest that the developmental pattern of locomotor sensitivity to ethanol is unique to the genotypic profile of the animal model.     

Differential neurosensitivity to the discriminative stimulus properties of ethanol in C57BL/6J and C3H/He mice

BACKGROUND: A large body of evidence suggests that the interoceptive cue associated with ethanol intoxication is complex and dependent on a number of environmental and biological factors. Despite the fact that mice have been widely used to study genetic influences on sensitivity to various actions of ethanol, few studies have used mice to examine sensitivity to the discriminative stimulus effects of ethanol. The purpose of this study was to compare sensitivity to the discriminative stimulus effects of ethanol in two inbred mouse strains, namely C57BL/6J and C3H/He mice. METHODS: Adult male C57BL/6J and C3H/He mice were trained to discriminate between ethanol and saline using a two-lever food reinforcement operant procedure. Once criterion discrimination performance was achieved, dose-response functions were determined from generalization tests. Additional experiments were conducted to determine whether differences in discrimination performance were related to differential blood/brain ethanol levels in the two mouse strains. RESULTS: A greater proportion of C57BL/6J mice acquired the discrimination and required fewer trials to achieve criterion performance compared with C3H/He mice with a 1.0 g/kg ethanol training dose. This deficit in acquisition was overcome when the training dose was increased to 2.0 g/kg for C3H/He mice. In a second experiment, a 1.5 g/kg training dose of ethanol was used for both strains. Again, a greater proportion of C57BL/6J mice acquired the discrimination and required fewer training trials to achieve criterion performance compared with C3H/He mice. Blood ethanol levels did not differ between the strains after administration of the 1.5 g/kg training dose. However, blood and brain ethanol levels did differ between the strains after doses of ethanol were administered that produced equivalent discrimination performance. CONCLUSIONS: Results indicate that ethanol discrimination was more readily acquired and maintained in C57BL/6J mice than C3H/He mice. Ethanol dose-response functions generated from generalization tests also clearly demonstrated greater sensitivity to the discriminative stimulus properties of ethanol in C57BL/6J mice compared with the C3H/He strain. This differential sensitivity to the interoceptive cue produced by ethanol does not seem to be related to learning or pharmacokinetic differences between the two inbred strains.     

Comparison of basal neuropeptide Y and corticotropin releasing factor levels between the high ethanol drinking C57BL/6J and low ethanol drinking DBA/2J inbred mouse strains

BACKGROUND: Recent genetic and pharmacological evidence indicates that low neuropeptide Y (NPY) levels in brain regions involved with neurobiological responses to ethanol promote increased ethanol consumption. Because of their opposing actions, it has been suggested that NPY and corticotropin releasing factor (CRF) exert a reciprocal regulation on drug self-administration. It has been widely reported that inbred C57BL/6 mice consume significantly higher amounts of ethanol than do DBA/2 mice. Therefore, we used immunohistochemical techniques to determine if basal NPY and/or CRF levels differed in predicted directions between C57BL/6J and DBA/2J mice. METHODS: Ethanol-naive C57BL/6J and DBA/2J mice were deeply anesthetized with sodium pentobarbital (100 mg/kg) and perfused transcardially with 0.1 mM of phosphate-buffered saline followed by 4% paraformaldehyde in buffered saline. Brains were collected and postfixed for 4 hr at 4 degrees C and then were cut into 35-microm sections. Tissues containing the nucleus accumbens (NAc), hypothalamus, and amygdala were processed for NPY or CRF immunoreactivity using immunofluorescent or DAB techniques. Immunoreactivity was quantified from digital images using Image J software. RESULTS: The C57BL/6J mice showed reduced NPY expression in the NAc shell, the basolateral amygdala, and the central nucleus of the amygdala when compared with DBA/2J mice. However, these strains did not differ in CRF expression in any of the brain regions analyzed. CONCLUSIONS: These data suggest that low NPY levels in the amygdala and/or the shell of the NAc, which are not compensated for by similar changes in CRF levels, may contribute to the high ethanol consumption characteristic of C57BL/6J mice.     

The Effects of Ethanol on Dopaminergic Neurons of the Ventral Tegmental Area Studied with Intracellular Recording in Brain Slices

Dopaminergic neurons in the ventral tegmental area of Tsai (VTA) have been implicated in the mediation of the rewarding effects of ethanol and many other drugs of abuse. Our previous extracellular studies in brain slices have demonstrated that ethanol increases the firing rate of dopaminergic neurons in the VTA. In the present intracellular study, ethanol (40–160 mM) increased the spontaneous firing rate of most (77%) VTA neurons. In addition, most (75%) VTA neurons were depolarized by ethanol. Ethanol also changed the shape of the spontaneous action potential in VTA neurons, reducing the amplitude of the spike after-hyperpolarization (in 74% of neurons) and also reducing the amplitude of the depolarizing phase of the action potential (in 86% of neurons tested). Furthermore, analysis of Voltage/ Current curves in the presence and absence of ethanol showed that ethanol had little effect on the resistance of the cell membrane at membrane potentials near rest, but enhanced the time-dependent inward rectification activated at more hyperpolarized membrane potentials (I_h). This intracellular study identifies several electrophysiological effects of ethanol that may underlie the ethanol-induced excitation of VTA neurons and, therefore, may be important for the rewarding effects of ethanol.     

Intravenous Ethanol Self-administration in C57BL/6J and DBA/2J Mice

Two strains of mice, C57BL/6J (B6) and DBA/2J (D2) were allowed to self-administer intravenous (iv) ethanol. These two strains were selected because they differ greatly in their preference for drinking ethanol solutions: 86 mice are preferrers, whereas D2 mice are avoiders of ethanol. Of interest was whether these strains would also differ in self-administration of iv ethanol when taste factors presumably do not influence consumption. Mice were trained with either 60, 75, or 90 mg/kg per infusion. Mice from both strains acquired nosepoking for all of these doses on an FR-3 schedule of reinforcement during 2-hr daily sessions. Additionally, mice in both strains acquired an equal preference for nosepoking on the side resulting in ethanol infusions, compared with the side that had no scheduled consequence, although B6 mice took somewhat more ethanol early in training than did D2 mice. Mice in both strains achieved equal levels of responding at the conclusion of training, when response rates had stabilized. A subset of animals were then tested at doses of ethanol ranging from 25 to 125 mg/kg per infusion. Although their responding tended to decrease over time regardless of changes in the unit dose of ethanol, these mice showed lower response rates for higher doses of ethanol, and less responding for saline than for ethanol. Together, these findings imply that iv ethanol has reinforcing properties in both these strains, despite the strain difference in preference for oral ethanol. Self-administration of iv ethanol in mice may prove a valuable addition to existing animal models for the study of ethanol reward.     

Analysis of metallothionein brain gene expression in relation to ethanol preference in mice using cosegregation and gene knockouts

BACKGROUND: Metallothioneins (MTs) are ubiquitously expressed intracellular proteins that bind heavy metals and are involved in cytoprotection against several types of stress agents including chemicals, hormones, and oxidants. We have previously reported 1 isoform, MT-II, as a possible candidate gene for ethanol (EtOH) preference (EP) determination in mice. METHODS: Semiquantitative RT-PCR was used to determine brain mRNA levels of MT-I and MT-III in 4 inbred mouse strains with variable EP. Following this, cosegregation of MT-II brain expression with EP was analyzed in F2 mice from 2 intercrosses (C57BL/6J x BALB/cJ and C57BL/6J x DBA/2J). Studies on MT-I/MT-II knockout (KO) mice were also undertaken to further explore this relationship. RESULTS: Our results suggest that MT-I is responsive to EtOH, with no evidence of basal-level differences between strains. Conversely, MT-III shows no EtOH response, yet indicates a possible strain-specific feature with C57BL/6J having the lowest levels of brain MT-III. Metallothionein-II expression cosegregates with EP in F2 mice from a C57BL/6J (preferring) and DBA/2J (avoiding) intercross. Although F2 mice from a cross with C57BL/6J and BALB/cJ (avoiding) strains follow a similar pattern, the results are not statistically significant. Metallothionein-I/MT-II knockout (MT-KO) mice appear to have smaller litter sizes as well as higher weight compared with controls (129S1/SvImJ) and also show a slight increase in EP. CONCLUSIONS: Metallothionein-II remains the primary candidate of the mouse MT gene family for involvement in EP. Its effect on EP appears to be dependent on the genetic background. Such conclusions are based on results from C57BL/6J, BALB/cJ, DBA/2J, and 129 inbred mouse strains. Evidence also points to shared neural pathways involved in weight gain and obesity. The complex interactions between MT-II, EP, and weight gain/obesity remain to be studied.     

Endogenous Opioids Are Involved in the Genetically Determined High Preference for Ethanol Consumption

The link between endogenous opioid peptides and the genetic predisposition to preferentially consume ethanol was examined in alcohol preferring C57BL/6J mice compared with the alcohol nonpreferring DBA/2 mice. Concentrations of Met-enkephalin pentapeptide or precursor in various brain regions of potential relevance were not different between the two strains. C57BL/6J mice had a significantly lower pain threshold that could be increased by a selective mu-receptor opioid agonist [D-Ala2, MePhe4, Met(O)5-ol]-enkephalin. Treatment with this drug also decreased ethanol consumption in C57BL/6J mice. Increasing the synaptic half-life of endogenous enkephalins by the enkephalinase inhibitor kelatorphan also decreased ethanol consumption. Assay of endogenous enkephalin degrading activity showed increased enkephalinase activity in striatal issue of C57BL/6J compared with DBA/2 tissue. These results suggest that a relative lack of enkephalin peptides trans-synaptically, possibly resulting from enhanced enkephalin degradation may contribute to increase alcohol consumption in C57BL/6J mice.     

Dietary-induced obesity and hypothalamic infertility in female DBA/2J mice

The effects of diet and adiposity have been implicated in disturbances of female reproductive function. In an effort to better elucidate the relationship between obesity and female fertility, we analyzed the effect of increasing dietary fat content on body composition, insulin sensitivity, and pregnancy rates in two common inbred mouse strains, DBA/2J and C57BL/6J. After 16 wk, females of both strains on the high fat diet developed glucose intolerance and insulin resistance, but only the female DBA/2J mice developed dietary-induced obesity and hyperleptinemia. The high fat diet was associated with more than a 60% decrease in natural pregnancy rates of female DBA/2J mice, whereas the fertility of female C57BL/6J mice was unaffected. Despite developing a similar degree of obesity, insulin resistance, and hyperleptinemia, male DBA/2J mice did not manifest diminished fertility. Obese female DBA/2J mice achieved normal ovulatory responses and pregnancy rates after exogenous gonadotropin stimulation, suggesting their fertility defect to be central in origin. Real-time PCR quantification of hypothalamic cDNA revealed a 100% up-regulation of neuropeptide Y and a 50% suppression of GnRH expression accompanied by a 95% attenuation of leptin receptor type B expression in obese female DBA/2J mice. These findings suggest that obesity-associated hyperleptinemia, and not insulin resistance or increased dietary fat per se, gradually induces central leptin resistance, increases hypothalamic neuropeptide Y-ergic tone, and ultimately causes hypothalamic hypogonadism. The data establish high fat-fed female DBA/2J mice as a wild-type murine model of obesity-related infertility.     

Role of Potassium Channel Gene Kcnj10 in Ethanol Preference in C57bl/6J and DBA/2J Mice

Background: Inwardly‐rectifying potassium channel protein Kir4.1 is encoded by Kcnj10 which maps to a quantitative trait locus on chromosome 1 for the voluntary alcohol consumption phenotype in mice. Kcnj10 brain expression differences have been established between ethanol‐preferring C57Bl/6J and ethanol‐avoiding BALB/cJ mice, but its differential expression in other tissues and strains have largely been overlooked. A nonsynonymous single nucleotide polymorphism exists between C57Bl/6J and ethanol‐avoiding DBA/2J mice which changes amino acid 262 from threonine (C57Bl/6J) to serine (DBA/2J). This Kcnj10 SNP and its expression may serve as valuable markers in predicting the ethanol preference phenotype in mice. Methods: The evolutionary divergence of the Kir gene family was characterized using phylogenetic analysis involving the 16 mouse Kir channels. Kcnj10 expression differences in the brain, liver, lung, heart, spleen, kidney, testes, and muscle of male C57Bl/6J and DBA/2J mice at different developmental stages were examined using semiquantitative RT‐PCR analysis. A SNP analysis was conducted to assess the association of Kcnj10 Thr262Ser SNP and the ethanol preference phenotype in F2 mice derived from the reciprocal crosses of the C57Bl/6J and DBA/2J strains. Results: Evolutionary analysis supports gene duplication and genetic recombination as likely sources of diversity within the Kir gene family. Semiquantitative RT‐PCR analysis revealed significantly higher Kcnj10 expression in the brain, spleen, and kidney of both strains when compared to other tissues from the same strain. There were no significant differences in tissue‐specific mRNA levels between strains except in the testes. Genotype distributions of the Kcnj10 Thr262Ser SNP were different between low‐ and high‐drinkers. A significant difference in the average ethanol preference level of each genotype was also observed. Conclusion: Our results suggest a role for Kcnj10 in ethanol preference determination in mice. However, further experiments are needed to establish if this association is due to the nonsynonymous SNP or other additional factors associated with Kcnj10.     

Brain region-specific regulation of urocortin 1 innervation and corticotropin-releasing factor receptor type 2 binding by ethanol exposure

BACKGROUND: Ethanol administration and consumption selectively activates the urocortin 1 (Ucn1)-expressing neurons of the Edinger-Westphal nucleus. We investigated whether repeated ethanol exposure affects Ucn1 and Ucn1-responsive corticotropin-releasing factor type-2 receptors (CRF2). METHODS: Male C57BL/6J and DBA/2J mice were exposed to 2 g/kg ethanol via intraperitoneal injection once per day for 14, seven, or zero days. Ucn1 immunoreactivity was measured in the lateral septum, dorsal raphe, and Edinger-Westphal nucleus. In a separate experiment, C57BL/6J mice were exposed to ethanol for seven, one, or zero days, and CRF2 receptor binding was measured in the lateral septum and dorsal raphe by receptor autoradiography. RESULTS: Ethanol exposure induced parallel changes in Ucn1 immunoreactive terminal fibers in the lateral septum and dorsal raphe of both strains. Seven ethanol exposures but not one ethanol exposure significantly increased CRF2 receptor binding in the dorsal raphe and slightly increased CRF2 receptor binding in the lateral septum. CONCLUSIONS: These results provide evidence that the Ucn1/CRF2 receptor system can be modified by ethanol exposure. They additionally suggest that this system may be involved in behavioral changes during alcoholism.     

Increased expression of hypothalamic leptin receptor and adiponectin accompany resistance to dietary-induced obesity and infertility in female C57BL/6J mice

BACKGROUND: Obesity is strongly associated with female infertility, but the mechanisms underlying this relationship are largely unknown. METHODS: We investigated the effect of increasing dietary fat percentage upon body mass, hypothalamic neuropeptide gene expression, adipose hormone secretion and fertility in females of the inbred mouse strains C57BL/6J and DBA/2J. To assess the effect of obesity independent of dietary influence, we also compared these parameters in wild-type female C57BL/6J mice to those congenic for the obesogenic mutations ob/ob and A(y)/a. RESULTS: After 24 weeks, rather than exhibiting an obese, leptin-resistant phenotype like their female DBA/2J counterparts, wild-type female C57BL/6J mice remained lean, fertile and manifested increased hypothalamic LEPR-B expression. Although both mutant genotypes were associated with obesity and subfertility, ob/ob mice demonstrated significantly increased hypothalamic LEPR-B expression, whereas A(y)/a mice had a significant reduction. Interestingly, wild-type female C57BL/6J mice were noted to manifest significantly higher and lower levels of adiponectin and tissue plasminogen activator inhibitor-1 (tPAI-1), respectively, than weight-matched wild-type female DBA/2J mice. CONCLUSIONS: We conclude that (1) resistance to the obese-infertile phenotype in female C57BL/6J mice is associated with increased hypothalamic leptin receptor expression and alterations in adipokine levels consistent with decreased adipose tissue inflammation and (2) that long-standing hyperleptinemic obesity in mice is associated with a downregulation of the hypothalamic leptin receptor.     

Comparative Pharmacokinetics of Ethanol in Inbred Strains of Mice Using Doses Based on Total Body Water

The mean total body water was determined by desiccation in DBA/2J, CBA/J, and C57BL/6J mice to be 60.6, 65.6, and 68.6 percent of body weight, respectively. The pharmacokinetics of ethanol was subsequently studied in mice of these strains given an intraperitoneal dose of 116 mmoles/l of total body water based on the desiccation study. This dose was equivalent to 70, 76, and 80 mmoles/kg in the DBA/2J, CBA/J, and C57BL/6J strains, respectively. The zero time concentrations were nearly identical between strains; therefore volume of distribution (VD) estimates based on mmole/kg doses reflected interstrain differences in total body water. The apparent zero order elimination rate was significantly greater in the DBA/2J strain versus the other two strains using this regimen. Interstrain differences in ethanol sleep time paralleled the differences in anesthetic sensitivity evidenced by blood concentrations at the time of regaining the righting reflex. The results demonstrate the importance of considering differences in total body water and hence ethanol VD when comparing the effects of ethanol in inbred mouse strains.     

Ethanol preference is inversely correlated with ethanol-induced dopamine release in 2 substrains of C57BL/6 mice

BACKGROUND: The C57BL/6 mouse model has been used extensively in alcohol drinking studies, yet significant differences in ethanol preference between substrains exist. Differences in ethanol-induced dopamine release in the ventral striatum could contribute to this variability in drinking behavior as dopamine has been implicated in the reinforcing properties of ethanol. METHODS: A 2-bottle choice experiment investigated the difference in ethanol preference between C57BL/6J and C57BL/6NCrl animals. Microdialysis was used to determine dopamine release and ethanol clearance in these 2 substrains after intraperitoneal injections of 1.0, 2.0 and 3.0 g/kg ethanol or saline. RESULTS: C57BL/6J mice exhibited significantly greater ethanol preference and less ethanol-stimulated dopamine release compared with C57BL/6NCrl mice. The intraperitoneal injections of ethanol caused a significant increase in dopamine in both substrains at all 3 doses with significant differences between substrains at the 2 highest alcohol doses. Saline injections had a significant effect on dopamine release when given in a volume equivalent to the 3 g/kg ethanol dose. Ethanol pharmacokinetics were similar in the 2 substrains at all 3 doses. CONCLUSIONS: Ethanol-induced dopamine release in the ventral striatum may contribute to the differences in alcohol preference between C57BL/6J and C57BL/6NCrl mice.     

Ethanol directly excites dopaminergic ventral tegmental area reward neurons

BACKGROUND: The mesolimbic/mesocortical dopamine pathway mediates the rewarding effects of ethanol and other drugs of abuse like cocaine and opiates. Dopaminergic neurons of the ventral tegmental area (VTA) are the cells of origin of the mesolimbic/mesocortical dopamine pathway. Ethanol's rewarding properties result from its ability to excite dopaminergic cell bodies in the VTA which results in increased dopamine release in the nucleus accumbens. Many recent papers have speculated that ethanol excitation of dopaminergic VTA neurons is indirect, either that ethanol acts on GABAergic or other interneurons, which in turn modulate the activity of dopaminergic VTA neurons, or that ethanol modulates the action of neurotransmitter-gated ion channels in the VTA. METHODS: VTA neurons were acutely dissociated and plated onto a cover slip in an electrophysiological recording chamber. These neurons generated spontaneous action potentials which could be measured with cell attached loose patch recording. The dissociation procedure truncated the dendritic trees, severed synaptic contacts and widely dispersed these neurons. Dopamine (10-50 nM) and ethanol (20-120 mM) were bath applied and their effects on firing rate were measured. After some experiments, plated cells were fixed and processed for immunostaining of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. RESULTS: All neurons met electrophysiological criteria previously established for dopaminergic VTA neurons. Dopamine inhibited all VTA neurons tested, indicating the presence of dopamine autoreceptors. All neurons identified as dopaminergic by these electrophysiological and pharmacological criteria, and that were processed for immunohistochemistry, stained positive for tyrosine hydroxylase immunoreactivity. All acutely dissociated VTA neurons, identified as dopaminergic by electrophysiological, pharmacological and immunohistochemical criteria, were robustly excited by behaviorally relevant concentrations of ethanol. The ethanol-induced excitation was concentration-dependent. CONCLUSIONS: These data provide strong evidence that ethanol directly excites dopaminergic VTA neurons, as this excitation still occurs in the absence of input from surrounding neurons.     

Acetaldehyde dehydrogenase (Ahd-2)-Associated DNA Polymorphisms in Mouse Strains with Variable Ethanol Preferences

The genotype-dependent response of mice to ethanol has been well documented. Cytosolic acetaldehyde dehydrogenase (ALDH-2) increases in some strains while decreasing in others with ethanol treatment. Further work suggests that the mRNA for ALDH-2 (Ahd-2 mRNA) levels are altered following ethanol feeding in a strain-dependent fashion. This report identifies differences in Ahd-2 at the genomic DNA level among different strains of mice. Restriction fragment length polymorphisms (RFLPs) associated with the Ahd-2 locus were found for the restriction enzymes EcoRI, HindIII, Pst I and Rsa I. The mouse strains included in this study could be categorized into two groups based on their overall Ahd-2 associated DNA banding patterns. Strains C57BL/6J, C57BL/6J*, C57BL/10J and BALB/c form group 1 while strains C3H/HeJ, C3H/HeSnJ, 129/ReJ, Csb, SW and DBA/2J form group 2. With the exception of BALB/c, group 1 represents alcohol preferring strains while group 2 are alcohol avoiding strains. Additional work will be required to determine the physiological significance (if any) of these RFLPs and their possible relationship to ethanol preference and avoidance.     

Alcohol, Cocaine, and Brain Stimulation-Reward in C57Bl6/J and DBA2/J Mice

Background: Pleasure and reward are critical features of alcohol drinking that are difficult to measure in animal studies. Intracranial self‐stimulation (ICSS) is a behavioral method for studying the effects of drugs directly on the neural circuitry that underlies brain reward. These experiments had 2 objectives: first, to establish the effects of alcohol on ICSS responding in the C57Bl6/J (C57) and DBA2/J (DBA) mouse strains; and second, to compare these effects to those of the psychostimulant cocaine. Methods: Male C57 and DBA mice were implanted with unipolar stimulating electrodes in the lateral hypothalamus and conditioned to spin a wheel for reinforcement by the delivery of rewarding electrical stimulation (i.e., brain stimulation‐reward or BSR). Using the curve‐shift method, the BSR threshold (θ₀) was determined immediately before and after oral gavage with alcohol (0.3, 0.6, 1.0, 1.7 g/kg) or water. Blood alcohol concentration (BAC) was measured to determine the influence of alcohol metabolism on BSR threshold. Separately, mice were administered cocaine (1.0, 3.0, 10.0, 30.0 mg/kg) or saline intraperitoneally. Results: In C57 mice, the 0.6 g/kg dose of alcohol lowered BSR thresholds by about 20%, during the rising (up to 40 mg/dl), but not falling, phase of BAC. When given to the DBA mice, alcohol lowered BSR thresholds over the entire dose range; the largest reduction was by about 50%. Cocaine lowered BSR thresholds in both strains. However, cocaine was more potent in DBA mice than in C57 mice as revealed by a leftward shift in the cocaine dose–response curve. For both alcohol and cocaine, effects on BSR threshold were dissociable from effects on operant response rates. Conclusions: In C57 and DBA mice, reductions in BSR threshold reflect the ability of alcohol to potentiate the neural mechanisms of brain reward. The DBA mice are more sensitive to the reward‐potentiating effects of both alcohol and cocaine, suggesting that there are mouse strain differences in the neural mechanisms of brain reward that can be measured with the ICSS technique.