Repeated Alcohol Administration Differentially Affects c-Fos and FosB Protein Immunoreactivity in DBA/2J Mice

To identify alcohol-responsive brain areas, we have immunohisto-chemically analyzed expression of c-Fos, FosB, and other Fos-related antigens in the brain of inbred DBN/2J mice after a single or repeated injection of alcohol (4 g/kg). We observed increased expression of c-Fos after alcohol administration in the central nucleus of amygdala, paraventricular nuclei of hypothalamus and thalamus, and several other brain areas. Although increased expression of c-Fos in the nucleus accumbens was also observed, this increase was not statistically significant. Repeated administration of alcohol had the tendency to reduce alcohol-induced c-Fos expression in these areas. Immunohistochemical analysis using an antibody recognizing most Fos-related antigens revealed increases of expression of these proteins in a partially overlapping set of brain regions. In contrast to c-Fos, FosB expression was found to be elevated significantly higher after repeated than after acute treatment with alcohol in several brain areas, including the shell of nucleus accumbens. In contrast to previous c-Fos studies, our studies confirm that alcohol administration indeed activates the reward circuits, including the basal ganglia, and suggest that FosB could serve as a more sensitive marker for this activation.     

Genetics, Ethanol and the Fos Response: A Comparison of the C57BL/6J and DBA/2J Inbred Mouse Strains

The effect of ethanol (0.25 to 4 g/kg) on the number of Fos-like immunoreactive (Fos-li) neurons was studied in the C57BL/6J (B6) and DBA/2J (D2) inbred mouse strains. The brain regions emphasized in the analysis were from the basal ganglia and some associated limbic nuclei. The question addressed was whether or not the D2 and B6 strains differed in these regions in a way that could explain the marked psychomotor stimulation of the D2, but not the B6, strain over the dose range of 1 to 2 g/kg of ethanol. Over the dose range of 0.25 to 2 g/kg, ethanol caused a modest increase in the number of Fos-li neurons within the caudate putamen (dorsolateral and dorsomedial) and the nucleus accumbens (core and shell), but there were no marked strain effects. There was no significant effect in either strain of ethanol treatment (0.25 to 2 g/kg) in the globus pallidus, ventral pallidum, and subthalamic nucleus. However, at 4 g/kg, there was a dramatic (>100%) increase of Fos-li neurons in the D2 but not B6 strain. A similar effect was noted in the entopeduncular nucleus, the substantia nigra zona reticulata (and compacta), but not the ventral tegmental area. A marked and substantial (>200%) Fos response was seen in the central amygdaloid nucleus (CeA) of the D2 strain over the entire dose range; in contrast, a substantial Fos response in the B6 strain was seen only at the 4 g/kg dose. The paraventricular thalamic nucleus, in general, paralleled data in the CeA; but, the Fos response was more modest, and the results for the D2 strain were significant only at the 2 g/kg dose. Overall, data suggest that ethanol at low to moderate doses induces significant, strain-dependent Fos responses in some limbic structures, but not in the basal ganglia. The possibility is considered that activation of some neurons in the CeA are permissive for expression of the ethanol-induced increase in motor activity.     

The impact of gonadectomy and adrenalectomy on acute withdrawal severity in male and female C57BL/6J and DBA/2J mice following a single high dose of ethanol

BACKGROUND: Steroid hormones can influence neuronal excitability and subsequent seizure susceptibility through genomic and nongenomic mechanisms. For example, there are proconvulsant steroids such as estradiol and corticosterone and anticonvulsant steroids such as testosterone, progesterone, and their GABAergic metabolites. Recent findings indicated that a single, acute administration of ethanol increased levels of GABAergic steroids and that the source of this increase was peripheral organs such as the adrenals and gonads. Thus, the purpose of the present study was to determine the impact of removal of the adrenals and/or gonads on withdrawal severity following a single high dose of ethanol in 2 genotypes that differ in ethanol withdrawal severity. METHOD: Male and female C57BL/6J (B6) and DBA/2J (D2) mice were either left intact (SHAM), adrenalectomized (ADX), gonadectomized (GDX), or underwent ADX/GDX surgery. Seven days following surgery, baseline handling-induced convulsions (HICs) were measured prior to administration of a 4 g/kg dose of ethanol. HICs were assessed following the ethanol injection, then hourly for 12 hours and at 24 hours. A separate group of mice were used to measure the impact of surgical status on ethanol metabolism at 30, 60, 120, and 240 minutes after a single 4 g/kg dose of ethanol. RESULTS: ADX and ADX/GDX treatments in male B6 and D2 mice increased ethanol withdrawal severity following a single dose of ethanol, measured by area under the withdrawal curve and peak HIC scores. Acute ethanol withdrawal also was increased in female D2 mice that had undergone ADX/GDX. In contrast, surgical status did not alter ethanol withdrawal severity in female B6 mice. Surgical status had only minor effects on ethanol metabolism. CONCLUSIONS: Removal of peripherally derived steroids with anticonvulsant properties significantly increased HIC scores during acute ethanol withdrawal following a single dose of ethanol in male and female D2 mice and in male B6 mice. These increases were not due to changes in ethanol metabolism.     

Chlordiazepoxide-Induced Expression of c-Fos in the Central Extended Amygdala and Other Brain Regions of the C57BL/6J and DBA/2J Inbred Mouse Strains: Relationships to Mechanisms of Ethanol Action

BACKGROUND: Previous studies have established an association between the ethanol-induced locomotor response and activation of the central nucleus of the amygdala (CeA) as determined by changes in the number of Fos-like immunoreactive (Fos-li) neurons. The purpose of the current study was to determine if the benzodiazepine chlordiazepoxide (CDP) would produce similar effects to ethanol on behavior and the CeA. METHODS: In experiment 1, C57BL/6J (B6) and DBA/2J (D2) mice were administered CDP over a dose range of 3 to 30 mg/kg and the effects on locomotor behavior and the number of Fos-li neurons in the extended CeA determined. For experiment 2, B6D2 F2 intercross animals were phenotyped for their locomotor response to ethanol using a test-retest design. The activity responsive and nonresponsive extreme phenotypes were then tested in the same fashion as the inbred strains. RESULTS: Similar to ethanol, CDP increased locomotor activity in the D2 strain but not the B6 strain; furthermore, the D2 strain was 3 to 10 times more sensitive than the B6 strain in terms of CDP activating the CeA and the associated lateral posterior aspect of the bed nucleus of the stria terminalis (BSTLP). In the shell of the nucleus accumbens (NAc), CDP inhibited the number of Fos-positive neurons in both strains. CDP also discriminated between the responsive and nonresponsive extremes both in terms of behavior and activation of the CeA. CONCLUSIONS: Overall, these data point to the importance of GABA(A) mediated mechanisms in the ethanol-induced locomotor response. It is suggested that both drugs block the feed-forward inhibition in the CeA, resulting in activation of the GABAergic projection neurons. The overall net effect of CDP or ethanol administration on the output from the CeA will be inhibitory, from which it follows that the locomotor activation response must be associated with the selective inhibition of some behavior or ensembles of behaviors that are known to be mediated by the CeA and reduce locomotor activity (i.e., the "freezing" response).     

Ethanol-induced expression of c-Fos differentiates the FAST and SLOW selected lines of mice

The effect of ethanol on the number of Fos-like immunoreactive (Fos-li) neurons was previously studied in the C57BL/6J (B6) and DBA/2J (D2) inbred mouse strains (Hitzemann and Hitzemann, 1997). Data obtained suggested that the locomotor activation response to ethanol found in the D2 but not the B6 strain was associated with an increase in the number of Fos-li neurons (a putative measure of synaptic activity) in the central nucleus of the amygdala (CeA), but not in other brain regions, including the basal ganglia. Supporting results were obtained in B6D2 F2 intercross animals (Demarest et al., 1998) those animals exhibiting a marked locomotor activation response to ethanol also showed a significant increase in the number of Fos-li neurons in the CeA. The current study extends this line of investigation to the FAST and SLOW selected lines of mice (Shen et al., 1995). Twenty-eight SLOW and FAST mice (taken evenly from both replicate lines) were randomly assigned to receive either saline or ethanol (1.5 g/kg). One hour later, the animals were sacrificed, and the number of Fos-li neurons were determined using standard immunocytochemical techniques. Both the FAST and SLOW lines showed a marked increase (>300%) in the number of Fos-li neurons in the lateral aspect of the CeA; however, in the capsular division, only the FAST line showed an increase (>500%). In several brain regions, the basal (saline) response was markedly higher in the SLOW line; these regions included the subthalamic nucleus, the entopeduncular nucleus, the substantia nigra compacta, and the ventral tegmental area. Furthermore, it was found that ethanol decreased the number of Fos-li neurons in the ventral tegmental area of the SLOW but not FAST mice. These data suggest a substantial involvement of the basal ganglia in the segregation of the FAST and SLOW lines.     

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.     

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.     

Chronic ethanol consumption by C57BL/6 mice promotes tolerance to its interoceptive cues and increases extracellular dopamine, an effect blocked by naltrexone

BACKGROUND: C57BL/6 (B6) mice voluntarily consume ethanol. Although preingestive factors might be accountable, the fact that B6 mice voluntarily consume sufficient ethanol to set the conditions for an ethanol-deprivation effect suggest that post-ingestive pharmacological induced changes also occur. In this study, we determined the amounts of ethanol voluntarily consumed by B6 mice and associated blood ethanol levels (BEL), the effects of this consumption on extracellular dopamine (DA) and how this was altered by naltrexone, as well as on its interoceptive discriminative cues. METHODS: In experiment 1, the amounts of 12% ethanol consumed at 2, 4, and 6 hr into the active phase of the circadian cycle and associated BEL were determined. In experiment 2, dialysate samples were collected for 1 hr to establish basal DA levels. Mice were then injected with saline or naltrexone (6 mg/kg) and given access to water and 12% ethanol or to water only, and samples were collected at 20-min intervals for the next 2 hr. In experiment 3, mice were trained to discriminate ethanol's interoceptive cues via operant techniques, and half were given 3 weeks access to ethanol and water, the other half water only. Ethanol-consuming and water control mice were again tested for their ability to discriminate the drug's interoceptive cues. RESULTS: Mice ingested nearly 6 g/kg of ethanol and attained BEL near 100 mg/100 mL by 6 hr into the active phase. Ethanol intake at 2-hr into the dark phase was approximately 2.5 g/kg, and increased DA to approximately 100% above basal levels. Naltrexone reduced ethanol consumption and blocked the DA increase. Ethanol consumption for 3 weeks attenuated its discriminative cues. CONCLUSIONS: B6 mice voluntarily consume sufficient ethanol (1) to produce intoxicating BEL; (2) to increase DA levels in nucleus accumbens, an effect blocked by naltrexone; and (3) to attenuate its discriminative cues.     

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.     

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.     

Effects of withdrawal from chronic intermittent ethanol vapor on the level and circadian periodicity of running-wheel activity in C57BL/6J and C3H/HeJ mice

Background: Alcohol withdrawal is associated with behavioral and chronobiological disturbances that may persist during protracted abstinence. We previously reported that C57BL/6J (B6) mice show marked but temporary reductions in running‐wheel activity, and normal free‐running circadian rhythms, following a 4‐day chronic intermittent ethanol (CIE) vapor exposure (16 hours of ethanol vapor exposure alternating with 8 hours of withdrawal). In the present experiments, we extend these observations in 2 ways: (i) by examining post‐CIE locomotor activity in C3H/HeJ (C3H) mice, an inbred strain characterized by high sensitivity to ethanol withdrawal, and (ii) by directly comparing the responses of B6 and C3H mice to a longer‐duration CIE protocol. Methods: In Experiment 1, C3H mice were exposed to the same 4‐day CIE protocol used in our previous study with B6 mice (referred to here as the 1‐cycle CIE protocol). In Experiment 2, C3H and B6 mice were exposed to 3 successive 4‐day CIE cycles, each separated by 2 days of withdrawal (the 3‐cycle CIE protocol). Running‐wheel activity was monitored prior to and following CIE, and post‐CIE activity was recorded in constant darkness to allow assessment of free‐running circadian period and phase. Results: C3H mice displayed pronounced reductions in running‐wheel activity that persisted for the duration of the recording period (up to 30 days) following both 1‐cycle (Experiment 1) and 3‐cycle (Experiment 2) CIE protocols. In contrast, B6 mice showed reductions in locomotor activity that persisted for about 1 week following the 3‐cycle CIE protocol, similar to the results of our previous study using a 1‐cycle protocol in this strain. Additionally, C3H mice showed significant shortening of free‐running period following the 3‐cycle, but not the 1‐cycle, CIE protocol, while B6 mice showed normal free‐running rhythms. Conclusions: These results reveal genetic differences in the persistence of ethanol withdrawal‐induced hypo‐locomotion. In addition, chronobiological alterations during extended abstinence may depend on both genetic susceptibility and an extended prior withdrawal history. The present data establish a novel experimental model for long‐term behavioral and circadian disruptions associated with ethanol withdrawal.     

Coregulation of ethanol discrimination by the nucleus accumbens and amygdala

BACKGROUND: Activation of GABA(A) receptors in the amygdala or nucleus accumbens produces discriminative stimulus effects that substitute fully for those of systemically administered ethanol. This study was conducted to determine if GABA(A) receptors in the amygdala and nucleus accumbens interactively modulate ethanol discrimination. METHODS: Male Long-Evans rats were trained to discriminate between intraperitoneal injections of ethanol (1 g/kg) and saline on a 2-lever drug discrimination task. The rats were then surgically implanted with bilateral injection cannulae aimed at the nucleus accumbens and the amygdala. RESULTS: Infusion of the GABA(A) agonist muscimol in the nucleus accumbens resulted in full substitution for systemically administered ethanol. Concurrent infusion of the GABA(A) antagonist bicuculline in the amygdala shifted the muscimol substitution curve in the nucleus accumbens 10-fold to the right. CONCLUSIONS: These results indicate that blockade of GABA(A) receptors in the amygdala significantly reduces the potency of the GABA(A) agonist in the nucleus accumbens. This suggests that the ethanol-like stimulus effects of GABA(A) receptor activation in the nucleus accumbens are modulated by GABA(A) receptor activity in the amygdala. These data support the hypothesis that the addictive stimulus properties of alcohol are mediated by GABAergic transmission in a neural circuit involving the amygdala and nucleus accumbens.     

Effect of ethanol on DARPP-32 phosphorylation in transgenic mice that express human type VII adenylyl cyclase in brain

BACKGROUND: Dopamine and cyclic adenosine monophosphate-regulated phosphoprotein of molecular weight 32 kDa (DARPP-32) is a bidirectional signaling protein found in dopaminergically innervated brain areas. The characteristics and direction of DARPP-32 effects are regulated by phosphorylation of this protein. Phosphorylation of DARPP-32 on threonine-34 (T34) is regulated through the activation of dopamine (D1) receptors and stimulation of adenylyl cyclase (AC) and protein kinase A activity and by calcineurin. Phosphorylation of DARPP-32 on threonine-75 (T75) is regulated by cyclin-dependent kinase 5 and protein phosphatase 2A. DARPP-32 has been implicated in the motivational effects of ethanol. METHODS: The authors characterized transgenic mice that overexpress an ethanol-sensitive isoform of AC (AC7) in brain by measuring basal and ethanol-modulated DARPP-32 phosphorylation. Phosphorylated and total DARPP-32 were measured by immunoblotting in brain areas associated with the motivational and anxiolytic effects of ethanol (nucleus accumbens, striatum, and amygdala). RESULTS: AC7 transgenic mice had higher basal levels of T34 DARPP-32 than wild-type mice in striatum and amygdala, whereas basal levels of T75 DARPP-32 did not differ between wild-type and transgenic mice. Ethanol administration increased T34 DARPP-32 in nucleus accumbens and amygdala (but not in the striatum) of wild-type and transgenic mice (with a greater effect in amygdala of transgenic mice than wild-type mice). Ethanol administration increased T75 DARPP-32 in amygdala of only the wild-type mice and in nucleus accumbens and striatum of both the transgenic and wild-type mice. CONCLUSIONS: The effect of ethanol on the balance of DARPP-32 phosphorylation, especially in amygdala of wild-type versus transgenic mice, may contribute to differential motivational effects of ethanol in these animals.     

Allelic variation in the GABA A receptor gamma2 subunit is associated with genetic susceptibility to ethanol-induced motor incoordination and hypothermia, conditioned taste aversion, and withdrawal in BXD/Ty recombinant inbred mice

BACKGROUND: Behavioral genomics has made dramatic progress toward mapping quantitative trait loci (QTLs) that contain genes responsible for phenotypic differences in a variety of behavioral responses to alcohol (ethanol). We previously identified a QTL on mouse Chromosome 11 that affects genetic predisposition to acute alcohol withdrawal. Among mice derived from the C57BL/6J (B6) and DBA/2J (D2) inbred strains, this QTL (Alcw3) accounts for 12% of the genetic variability in withdrawal liability. Candidate genes within this QTL encode the gamma-aminobutyric acid type A (GABA A) receptor gamma2, alpha1, alpha6, and beta2 subunits. We recently identified a coding sequence polymorphism between the B6 and D2 strains for the GABA A receptor gamma2 subunit gene (Gabrg2). In this study, we expand our analysis to a panel of BXD strains derived from the B6 and D2 progenitor strains. These BXD strains provide 26 fixed recombinant genotypes that can be used to examine genetic correlations, for example, between a phenotype of interest and allelic variation in a candidate gene. METHODS: Gabrg2 was cloned and sequenced from the 26 BXD recombinant inbred strains. We analyzed genetic correlations between allelic variation in Gabrg2 and alcohol phenotypes previously measured in the BXD strain means. RESULTS: Allelic variation in Gabrg2 is correlated genetically with predisposition to acute alcohol withdrawal and may underlie the Alcw3 locus. In addition, Gabrg2 is associated with ethanol-conditioned taste aversion, ethanol-induced motor incoordination, and ethanol-induced hypothermia. A trend is observed for chronic ethanol withdrawal, ethanol-induced loss of righting reflex, and tolerance to ethanol-induced hypothermia and ataxia. CONCLUSIONS: Functionally relevant variation in Gabrg2, or a closely linked gene, is correlated genetically with some, but not all, behavioral responses to alcohol. The alcohol-related phenotypes associated with Gabrg2 generally may be characterized as debilitating or motivationally negative.     

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.     

Further Evidence That the Central Nucleus of the Amygdala Is Associated with the Ethanol-Induced Locomotor Response

The effect of ethanol on the number of Fos-like immunoreactive (Fos-li) neurons was previously studied in the C57BUW (B6) and DBA/2J (D2) inbred mouse strains (Hitzemann and Hitzemann, Alcohol. Clin. Exp. Res. 21:1497–1507, 1997). The data obtained suggested that the locomotor activation response to ethanol found in the D2 but not the B6 strain was associated with an increase in the number of Fos-li neurons (a putative measure of synaptic activity) in the central nucleus of the amygdala (CeA), but not in other brain regions, including the basal ganglia. The current study was performed to obtain data supporting a role for the CeA in the locomotor response. B6D2 F₂ intercross animals were phenotyped for their locomotor response to ethanol (1.5 g/kg). The animals from the extreme phenotypes (> 1 SD from the mean) were denoted as very high and very low activity (HH, LL) and differed in their ethanol response by >9,000 cm/15 min (baseline activity was similar in both phenotypes: 5,500 cm/15 min). These extremes especially differed from the parental strains in that the LL group showed a significant ethanol-induced inhibition of activity. After 2 weeks, HH and LL animals were rechallenged with 1.5 g/kg of ethanol or saline and the number of Fos-li neurons determined 1 hr later. In the HH group, ethanol increased the number of Fos-li neurons >600%, whereas in the LL group the increase was 170% (difference: p < 0.001). The increase in the HH group was principally located in the GABA neuron-rich lateral aspect of the CeA and not in the medial posterior-ventral division or the caps division. No significant difference was found between groups in the Fos response for the basolateral or lateral amygdala. Other brain regions were also examined, including the basal ganglia, the hippocampus (CA1, CA3, and dentate gyms), the bed nucleus of the stria terminalis, and several cortical regions. In some regions (e.g., the bed nucleus), a significant ethanol effect was detected, but it did not differentiate the HH and LL groups. Overall, the data obtained further argue that the CeA has an important role in regulating the acute locomotor response to ethanol.     

Content of Dynorphins and k-Opioid Receptors in Distinct Brain Regions of C57BL/6 and DBA/2 Mice

Differences in the activity of various components of the endogenous opioid system under basal conditions and after ethanol exposure have been reported between strains and lines of animals showing either high or low ethanol consumption. The objective of the present studies was to investigate the presence of differences in (a) the density of k-opioid binding sites, (b) the content of prodynorphin mRNA, and (c) the content of dynorphin peptides in distinct brain regions between the C57BL/6 (ethanol-preferring) and the DBA/2 (ethanol-avoiding) mice. Results indicated that the C57BL/6 mice have a higher content of k-opioid binding sites and dynorphin A1-13 in the amygdala, and dynorphin A 1–8 in the ventral tegmental area, whereas the DBA/2 mice presented a significantly higher content of k-opioid binding sites, prodynorphin mRNA, as well as dynorphin A 1–13 and dynorphin A 1–8 peptides in the nucleus accumbens and septum. In addition, the DBA/2 mice presented a higher content of k-opioid receptors in the periaqueductal gray and dynorphin A1-13 and dynorphin A 1–8 in the caudate putamen. Because enhanced stimulation of the k-opioid receptors at the level of the nucleus accumbens has been associated with decreased dopamine release and aversive states, the higher content of k-opioid receptors, prodynorphin mRNA, and dynorphin peptides (the endogenous ligand of k-binding sites) in regions of the limbic system of the DBA/2 mice may play an important role in determining their low alcohol consumption.     

Persistent escalation of alcohol drinking in C57BL/6J mice with intermittent access to 20% ethanol

Background: Intermittent access (IA) to drugs of abuse, as opposed to continuous access, is hypothesized to induce a kindling‐type transition from moderate to escalated use, leading to dependence. Intermittent 24‐hour cycles of ethanol access and deprivation can generate high levels of voluntary ethanol drinking in rats. Methods: The current study uses C57BL/6J mice (B6) in an IA to 20% ethanol protocol to escalate ethanol drinking levels. Adult male and female B6 mice were given IA to 20% ethanol on alternating days of the week with water available ad libitum. Ethanol consumption during the initial 2 hours of access was compared with a short‐term, limited access “binge” drinking procedure, similar to drinking‐in‐the‐dark (DID). B6 mice were also assessed for ethanol dependence with handling‐induced convulsion, a reliable measure of withdrawal severity. Results: After 3 weeks, male mice given IA to ethanol achieved high stable levels of ethanol drinking in excess of 20 g/kg/24 h, reaching above 100 mg/dl blood ethanol concentrations, and showed a significantly higher ethanol preference than mice given continuous access to ethanol. Also, mice given IA drank about twice as much as DID mice in the initial 2‐hour access period. B6 mice that underwent the IA protocol for longer periods of time displayed more severe signs of alcohol withdrawal. Additionally, female B6 mice were given IA to ethanol and drank significantly more than males (ca. 30 g/kg/24 h). Discussion: The IA method in B6 mice is advantageous because it induces escalated, voluntary, and preferential per os ethanol intake, behavior that may mimic a cardinal feature of human alcohol dependence, though the exact nature and site of ethanol acting in the brain and blood as a result of IA has yet to be determined.     

Influence of early-life adversity on responses to acute and chronic ethanol in female mice

Background

Stressful early-life experiences increase the risk of developing an alcohol use disorder. We previously found that male C57BL/6J mice reared under limited bedding and nesting (LBN) conditions, a model of early-life adversity, escalate their ethanol intake in limited-access two-bottle choice (2BC) sessions faster than control (CTL)-reared counterparts when exposed to chronic intermittent ethanol (CIE) vapor inhalation. However, the alcohol consumption of female littermates was not affected by LBN or CIE. In the present study, we sought to determine whether this phenotype reflected a general insensitivity of female mice to the influence of early-life stress on alcohol responses.

Methods

In a first experiment, CTL and LBN females with a history of 2BC combined or not with CIE were tested in affective and nociceptive assays during withdrawal. In a second group of CTL and LBN females, we examined ethanol-induced antinociception, sedation, plasma clearance, and c-Fos induction.

Results

In females withdrawn from chronic 2BC, CIE increased digging, reduced grooming, and increased immobility in the tail suspension test regardless of early-life history. In contrast, LBN rearing lowered mechanical nociceptive thresholds regardless of CIE exposure. In females acutely treated with ethanol, LBN rearing facilitated antinociception and delayed the onset of sedation without influencing ethanol clearance rate or c-Fos induction in the paraventricular nucleus of the hypothalamus, paraventricular nucleus of the thalamus, central nucleus of the amygdala, or auditory cortex.

Conclusion

CIE withdrawal produced multiple indices of negative affect in C57BL/6J females, suggesting that their motivation to consume alcohol may differ from air-exposed counterparts despite equivalent intake. Contrasted with our previous findings in males, LBN-induced mechanical hyperalgesia in chronic alcohol drinkers was specific to females. Lower nociceptive thresholds combined with increased sensitivity to the acute antinociceptive effect of ethanol may contribute to reinforcing ethanol consumption in LBN females but are not sufficient to increase their intake.     

Adolescent C57BL/6J (but not DBA/2J) Mice Consume Greater Amounts of Limited‐Access Ethanol Compared to Adults and Display Continued Elevated Ethanol Intake into Adulthood

Background: Alcohol use is common during the adolescent period, a time at which a number of crucial neurobiological, hormonal, and behavioral changes occur ( Spear, 2000 ). In order to more fully understand the complex interaction between alcohol use and these age‐typical neurobiological changes, animal models must be utilized. Rodents experience a developmental period similar to that of adolescence. Although rat models have shown striking adolescent‐specific differences in sensitivity to ethanol, little work has been done in mice despite the fact that the C57BL/6J (B6) and DBA2/J (D2) mice have been shown to markedly differ in ethanol preference drinking and exhibit widely different sensitivities to ethanol. Methods: The current study examined ethanol intake in adolescent and adult B6 and D2 mice using a limited access alcohol exposure paradigm called Drinking in the Dark (DID). Additionally, the effect of adolescent (or adult) ethanol exposure on subsequent adult ethanol intake was examined by re‐exposing the mice to the same paradigm once the adolescents reached adulthood. We hypothesized that adolescent (P25–45) mice would exhibit greater binge‐like alcohol intake compared to adults (P60–80), and that B6 mice would exhibit greater binge‐like alcohol intake compared to D2 mice. Moreover, we predicted that relative difference in binge‐like alcohol intake between adolescents and adults would be greater in D2 mice. Results: Adolescent B6 mice consumed more ethanol than adults in the DID model. There was no difference between adolescent and adult D2 mice. Conclusions: This work adds to the literature suggesting that adolescents will consume more ethanol than adults and that this exposure can result in altered adult intake. However, this effect seems largely dependent upon genotype. Future work will continue to examine age‐related differences in ethanol intake, preference, and sensitivity in inbred mouse strains.