Escalating caffeine dose-dependently increases alcohol consumption in adult male, but not female, C57BL/6J mice.

Although previous research has illustrated the effects of the consumption of alcohol and caffeine individually, less research has focused on the popular combination of the two drugs. The increase in alcohol consumption when combined with caffeine has led to the idea that the stimulant effects of caffeine may mask the depressant effects of alcohol, and this may contribute to increased binge drinking as the individual feels more awake and stimulated. Preclinical research has shown various effects of combined alcohol and caffeine where several studies show decreased alcohol consumption and others show increased alcohol consumption and even binge-like drinking. Results from a previous study in our lab indicate that intermittent access (IA) to steady levels of low (0.015 %) but not moderate (0.03 %) caffeine increased alcohol consumption in male C57BL/6J mice. The current studies further investigated the sex and dose differences in adult mice receiving varying concentrations of caffeine on combined alcohol intake. In Experiment 1, adult mice (n = 50, 25 males and 25 females) had IA to one of the following experimental bottles throughout the 4 week period: water, alcohol (10 % v/v), caffeine (0.015 % w/v), or 10 % alcohol +0.015 % caffeine. In Experiment 2, adult mice (n = 70, 35 males and 35 females) were given IA to one of the following experimental bottles: water, alcohol (10 % v/v; steady, maintained throughout the 4 weeks), caffeine (increasing 0.01 % to 0.015 % to 0.02 % to 0.03 % weekly), or 10 % alcohol+increasing caffeine (at the previously mentioned concentrations). When both caffeine and alcohol concentrations remained steady throughout the 4 weeks, there was no change in alcohol consumption. Chronic exposure to IA caffeine led to increased locomotor activity and decreased freezing episodes when tested in the open field test approximately 6 h after removal of the bottles. In Experiment 2, caffeine dose-dependently increased alcohol co-consumption in male mice whereas female mice consumed less alcohol when it was presented in conjunction with caffeine. The results in males are in line with clinical literature suggesting that the combination of alcohol and caffeine may lead to increased stimulation and alcohol drinking. Additionally, these studies provide evidence that the escalation of caffeine is crucial when investigating alcohol and caffeine co-consumption using the IA paradigm.

Low but not moderate amounts of caffeine increase co-consumption of ethanol in C57BL/6J mice.

The increasingly popular combination of "energy drinks" containing high amounts of caffeine and alcohol has been shown to induce a stimulated, rather than sedated, state which may result in increased binge drinking and increased risk for alcohol-attributable accidents. We sought to examine consumption patterns of and withdrawal from alcohol and caffeine using a voluntary co-consumption animal model. Male and female adult C57BL/6J mice were given access to increasing doses of caffeine (0.01-0.05%) and/or alcohol (3-20%) in a two-bottle choice, intermittent access voluntary paradigm with fluid consumption recorded daily. Anxiety-like behavior during withdrawal was assessed via elevated plus maze or open field test in experiment 2. Increasing both alcohol and caffeine simultaneously in Experiment 1 resulted in no significant changes in co-consumption compared to mice given access to only alcohol or caffeine. Experiment 2 held caffeine concentration steady while slowly increasing alcohol content and resulted in mice consuming more alcohol when it was consumed in tandem with low dose caffeine. Both male and female mice consumed more caffeine when it was paired with alcohol; however, no significant differences were observed during withdrawal behavior. These results suggest that caffeine may dose-dependently positively influence alcohol consumption in mice and echo clinical literature suggesting that caffeine and alcohol together may result in a heightened state of stimulation and lead to further binge drinking. The intermittent access paradigm affords increased translational validity regarding investigations of alcohol and caffeine co-consumption and may be useful in identifying the neurobiological mechanisms concerning co-consumption of such substances.

Gene editing vectors for studying nicotinic acetylcholine receptors in cholinergic transmission.

Nicotinic acetylcholine receptors (nAChRs), prototype members of the cys-loop ligand-gated ion channel family, are key mediators of cholinergic transmission in the central nervous system. Despite their importance, technical gaps exist in our ability to dissect the function of individual subunits in the brain. To overcome these barriers, we designed CRISPR/Cas9 small guide RNA sequences (sgRNAs) for the production of loss-of-function alleles in mouse nAChR genes. These sgRNAs were validated in vitro via deep sequencing. We subsequently targeted candidate nAChR genes in vivo by creating herpes simplex virus (HSV) vectors delivering sgRNAs and Cas9 expression to mouse brain. The production of loss-of-function insertions or deletions (indels) by these 'all-in-one' HSV vectors was confirmed using brain slice patch clamp electrophysiology coupled with pharmacological analysis. Next, we developed a scheme for cell type-specific gene editing in mouse brain. Knockin mice expressing Cas9 in a Cre-dependent manner were validated using viral microinjections and genetic crosses to common Cre-driver mouse lines. We subsequently confirmed functional Cas9 activity by targeting the ubiquitous neuronal protein, NeuN, using adeno-associated virus (AAV) delivery of sgRNAs. Finally, the mouse ß2 nAChR gene was successfully targeted in dopamine transporter (DAT)-positive neurons via CRISPR/Cas9. The sgRNA sequences and viral vectors, including our scheme for Cre-dependent gene editing, should be generally useful to the scientific research community. These tools could lead to new discoveries related to the function of nAChRs in neurotransmission and behavioral processes.

Behavioral Characterization of ß-Arrestin 1 Knockout Mice in Anxiety-Like and Alcohol Behaviors.

ß-Arrestin 1 and 2 are highly expressed proteins involved in the desensitization of G protein-coupled receptor signaling which also regulate a variety of intracellular signaling pathways. Gene knockout (KO) studies suggest that the two isoforms are not homologous in their effects on baseline and drug-induced behavior; yet, the role of ß-arrestin 1 in the central nervous system has been less investigated compared to ß-arrestin 2. Here, we investigate how global ß-arrestin 1 KO affects anxiety-like and alcohol-related behaviors in male and female C57BL/6 mice. We observed increased baseline locomotor activity in ß-arrestin 1 KO animals compared with wild-type (WT) or heterozygous (HET) mice with a sex effect. KO male mice were less anxious in a light/dark transition test, although this effect may have been confounded by increased locomotor activity. No differences in sucrose intake were observed between genotypes or sexes. Female ß-arrestin 1 KO mice consumed more 10% alcohol than HET females in a limited 4-h access, two-bottle choice, drinking-in-the-dark model. In a 20% alcohol binge-like access model, female KO animals consumed significantly more alcohol than HET and WT females. A significant sex effect was observed in both alcohol consumption models, with female mice consuming greater amounts of alcohol than males relative to body weight. Increased sensitivity to latency to loss of righting reflex (LORR) was observed in ß-arrestin 1 KO mice although no differences were observed in duration of LORR. Overall, our efforts suggest that ß-arrestin 1 may be protective against increased alcohol consumption in females and hyperactivity in both sexes.

Broad-spectrum protein kinase inhibition by the staurosporine analog KT-5720 reverses ethanol withdrawal-associated loss of NeuN/Fox-3.

Chronic, intermittent ethanol (CIE) exposure is known to produce neuroadaptive alterations in excitatory neurotransmission that contribute to the development of dependence. Although activation of protein kinases (e.g., cyclic AMP [cAMP]-dependent protein kinase) is implicated in the synaptic trafficking of these receptors following CIE exposure, the functional consequences of these effects are yet to be fully understood. The present study sought to delineate the influence of protein kinase in regulating cytotoxicity following CIE exposure, as well as to examine the relative roles of ethanol exposure and ethanol withdrawal (EWD) in promoting these effects. Rat hippocampal explants were exposed to a developmental model of CIE with or without co-application of broad-spectrum protein kinase inhibitor KT-5720 (1 µM) either during ethanol exposure or EWD. Hippocampal cytotoxicity was assessed via immunofluorescence (IF) of neuron-specific nuclear protein (NeuN) with thionine staining of Nissl bodies to confirm IF findings. Concomitant application of ethanol and KT-5720 restored the loss of NeuN/Fox-3 IF in pyramidal CA1 and granule DG cell layers produced by CIE, but there was no restoration in CA3. Application of KT-5720 during EWD failed to significantly alter levels of NeuN IF, implying that ethanol exposure activates protein kinases that, in part, mediate the effects of EWD. KT-5720 application during EWD also restored thionine staining in CA1, suggesting kinase regulation of both neurons and non-neuronal cells. These data demonstrate that CIE exposure alters protein kinase activity to promote ethanol withdrawal-associated loss of NeuN/Fox-3 and highlight the influence of kinase signaling on distinct cell types in the developing hippocampus.

Altered nicotine reward-associated behavior following α4 nAChR subunit deletion in ventral midbrain.

Nicotinic acetylcholine receptors containing α4 subunits (α4ß2* nAChRs) are critical for nicotinic cholinergic transmission and the addictive action of nicotine. To identify specific activities of these receptors in the adult mouse brain, we coupled targeted deletion of α4 nAChR subunits with behavioral and and electrophysiological measures of nicotine sensitivity. A viral-mediated Cre/lox approach allowed us to delete α4 from ventral midbrain (vMB) neurons. We used two behavioral assays commonly used to assess the motivational effects of drugs of abuse: home-cage oral self-administration, and place conditioning. Mice lacking α4 subunits in vMB consumed significantly more nicotine at the highest offered nicotine concentration (200 µg/mL) compared to control mice. Deletion of α4 subunits in vMB blocked nicotine-induced conditioned place preference (CPP) without affecting locomotor activity. Acetylcholine-evoked currents as well as nicotine-mediated increases in synaptic potentiation were reduced in mice lacking α4 in vMB. Immunostaining verified that α4 subunits were deleted from both dopamine and non-dopamine neurons in the ventral tegmental area (VTA). These results reveal that attenuation of α4* nAChR function in reward-related brain circuitry of adult animals may increase nicotine intake by enhancing the rewarding effects and/or reducing the aversive effects of nicotine.

Corticosterone enhances N-methyl-D-aspartate receptor signaling to promote isolated ventral tegmental area activity in a reconstituted mesolimbic dopamine pathway.

Elevations in circulating corticosteroids during periods of stress may influence activity of the mesolimbic dopamine reward pathway by increasing glutamatergic N-methyl-D-aspartate (NMDA) receptor expression and/or function in a glucocorticoid receptor-dependent manner. The current study employed organotypic co-cultures of the ventral tegmental area (VTA) and nucleus accumbens (NAcc) to examine the effects of corticosterone exposure on NMDA receptor-mediated neuronal viability. Co-cultures were pre-exposed to vehicle or corticosterone (CORT; 1 µM) for 5 days prior to a 24 h co-exposure to NMDA (200 µM). Co-cultures pre-exposed to a non-toxic concentration of corticosterone and subsequently NMDA showed significant neurotoxicity in the VTA only. This was evidenced by increases in propidium iodide uptake as well as decreases in immunoreactivity of the neuronal nuclear protein (NeuN). Co-exposure to the NMDA receptor antagonist 2-amino-7-phosphonovaleric acid (APV; 50 µM) or the glucocorticoid receptor (GR) antagonist mifepristone (10 µM) attenuated neurotoxicity. In contrast, the combination of corticosterone and NMDA did not produce any significant effects on either measure within the NAcc. Cultures of the VTA and NAcc maintained without synaptic contact showed no response to CORT or NMDA. These results demonstrate the ability to functionally reconstitute key regions of the mesolimbic reward pathway ex vivo and to reveal a GR-dependent enhancement of NMDA receptor-dependent signaling in the VTA.

Ethanol withdrawal is required to produce persisting N-methyl-D-aspartate receptor-dependent hippocampal cytotoxicity during chronic intermittent ethanol exposure.

Chronic intermittent ethanol consumption is associated with neurodegeneration and cognitive deficits in preclinical laboratory animals and in the clinical population. While previous work suggests a role for neuroadaptations in the N-methyl-d-aspartate (NMDA) receptor in the development of ethanol dependence and manifestation of withdrawal, the relative roles of ethanol exposure and ethanol withdrawal in producing these effects have not been fully characterized. To examine underlying cytotoxic mechanisms associated with chronic intermittent ethanol (CIE) exposure, organotypic hippocampal slices were exposed to 1-3 cycles of ethanol (50 mM) in cell culture medium for 5 days, followed by 24 h of ethanol withdrawal, in which a portion of slices were exposed to competitive NMDA receptor antagonist (2R)-amino-5-phosphonovaleric acid (APV; 40 µM). Cytotoxicity was assessed using immunohistochemical labeling of neuron-specific nuclear protein (NeuN; Fox-3), a marker of mature neurons, and thionine (2%) staining of Nissl bodies. Multiple cycles of CIE produced neurotoxicity, as reflected in persisting losses of neuron NeuN immunoreactivity and thionine staining in each of the primary cell layers of the hippocampal formation. Hippocampi aged in vitro were significantly more sensitive to the toxic effects of multiple cycles of CIE than were non-aged hippocampi. This effect was not demonstrated in slices exposed to continuous ethanol, in the absence of withdrawal, or to a single exposure/withdrawal regimen. Exposure to APV significantly attenuated the cytotoxicity observed in the primary cell layers of the hippocampus. The present findings suggest that ethanol withdrawal is required to produce NMDA receptor-dependent hippocampal cytotoxicity, particularly in the aging hippocampus in vitro.

Long-term ethanol and corticosterone co-exposure sensitize the hippocampal ca1 region pyramidal cells to insult during ethanol withdrawal in an NMDA GluN2B subunit-dependent manner.

BACKGROUND: Chronic ethanol (EtOH) exposure produces neuroadaptations in NMDA receptor function and/or abundance and alterations in hypothalamic-pituitary-adrenal (HPA) axis functioning that contribute to neuronal excitation and neurotoxicity during ethanol withdrawal (EWD). Both EtOH and corticosterone (CORT) promote synthesis of polyamines, which allosterically potentiate NMDA receptor function at the GluN2B subunit. The current studies investigated the effect of 10-day EtOH and CORT co-exposure on toxicity during EWD in rat hippocampal explants and hypothesized that alterations in function and/or density of GluN2B subunits contribute to the toxicity. METHODS: Organotypic hippocampal slice cultures were exposed to CORT (0.01-1.0 µM) during 10-day EtOH exposure (50 mM) and 1 day of EWD. EtOH-naïve cultures were exposed to CORT for 11 days. Additional cultures were exposed to a membrane impermeable form of CORT (BSA-CORT) with and without 10-day EtOH exposure and EWD. Cytotoxicity (uptake of propidium iodide) was assessed in the pyramidal cell layer of the CA1 region. Western blot analysis was employed to assess the density of GluN2B subunits following EtOH and CORT exposure. RESULTS: EWD did not produce overt neurotoxicity. However, co-exposure to EtOH/EWD and CORT produced significant neurotoxicity in the CA1 region pyramidal cell layer. Ifenprodil, a GluN2B polyamine site antagonist, significantly reduced toxicity from EtOH and CORT (0.1 µM) co-exposure during EWD. However, Western blots did not reveal differences in GluN2B subunit density among groups. Exposure to BSA-CORT did not produce toxicity, suggesting that membrane-bound CORT receptors did not significantly contribute to the observed toxicity. CONCLUSIONS: These data suggest that CORT and EtOH co-exposure result in increased function of polyamine-sensitive GluN2B subunits, but this toxicity does not appear dependent on the abundance of hippocampal NMDA GluN2B subunits or membrane-bound CORT receptor function.

Sex differences in neuroadaptation to alcohol and withdrawal neurotoxicity.

Recent work suggests that sex differences exist with regard to both the nature of neuroadaptation to alcohol during the development of dependence, and possibly, the neurodegenerative consequences of alcohol dependence. Volumetric studies in human samples show that females may demonstrate increased volumetric brain loss with equal or lesser dependence histories than males. Furthermore, animal studies demonstrate sex differences in glutamatergic, GABAergic, and adenosinergic receptor signaling and endocrine responses following prolonged alcohol exposure. These differences may influence the development of dependence, neuronal function, and viability, particularly during alcohol withdrawal. The present review discusses the current state of knowledge in this regard. It is concluded that there exists a clear need for a more extensive examination of potential sex differences in neurodegenerative consequences of alcohol dependence in men and women, particularly with regard to the role that alterations in amino acid signaling and hypothalamic-pituitary-adrenal axis function may play. Furthermore, we note the need for expanded examination of the unique role that alcohol withdrawal-associated neuronal activity may have in the development of dependence-associated neurotoxicity.

Mifepristone pretreatment reduces ethanol withdrawal severity in vivo.

BACKGROUND: Prolonged ethanol (EtOH) intake may perturb function of the hypothalamic-pituitary-adrenal axis in a manner that promotes dependence and influences EtOH withdrawal severity. Prior in vivo and in vitro studies suggest that corticosteroids, in particular, may be elevated during EtOH intoxication and withdrawal, suggesting that intracellular glucocorticoid receptors (GRs) may promote the development of EtOH dependence. METHODS: Adult male Sprague-Dawley rats were subjected to a 4-day binge-like EtOH administration regimen (3 to 5 g/kg/i.g. every 8 hours designed to produce peak blood EtOH levels (BELs) of <300 mg/dl). Subgroups of animals received s.c. injection of the GR antagonist mifepristone (20 or 40 mg/kg in peanut oil at 0800 hours on each of the 4 days prior to withdrawal). BELs were assessed at 0900 and 1500 hours on Days 2 (D2) and 4 (D4) of the regimen. BEL, blood corticosterone levels (BCLs), and EtOH withdrawal-associated behavioral abnormalities were assessed 10 to 12 hours after the final EtOH administration. RESULTS: Daily mean EtOH doses for D1 to D4 of the regimen were 14.4, 9.9, 7.1, and 8.6 g/kg, respectively. The EtOH gavage regimen produced mean BELs of 255 mg/dl at 0900 on D2 and 156.2 mg/dl at 0900 on D4 of the regimen. Withdrawal from the EtOH exposure regimen, beginning 10 hours after the last EtOH administration, produced significant elevations in BCL and behavioral abnormalities including tremors, stereotypy, and "wet dog shakes." Mifepristone administration did not alter food intake or weight during the 4-day regimen, nor were there drug-dependent differences in BEL or BCL on withdrawal day. Although mifepristone produced no significant changes in behavior of EtOH-naïve animals, pretreatment with mifepristone (40 mg/kg) significantly reduced the severity of EtOH withdrawal. CONCLUSIONS: Findings suggest that activation of GRs promotes neuroadaptation to binge-like EtOH exposure, contributing to the development of EtOH dependence. Further, GRs may represent therapeutic targets to be exploited in reducing the severity of EtOH withdrawal.

Reinstatement of methamphetamine conditioned place preference in nicotine-sensitized rats.

The current experiments examined the effects of repeated nicotine prior to acquisition, extinction, and reinstatement of methamphetamine-induced conditioned place preference (CPP). Methamphetamine-induced (METH; 0.25, 0.5, or 1 mg/kg, s.c.) CPP was established using separate groups of adult male Sprague-Dawley rats with an unbiased conditioning procedure. Following extinction of METH CPP, drug-primed reinstatement (0, 0.25, 0.5 or 1 mg/kg, s.c.) of METH CPP was assessed in order to determine whether METH-induced reinstatement depends on the METH dose used to induce CPP. In a second experiment, separate groups of rats received nicotine (NIC; 0 or 0.2 mg/kg, s.c.) for 7 days prior to undergoing METH (0 or 0.5 mg/kg, s.c.) conditioning, extinction, and drug-primed reinstatement. Results indicate that METH-primed reinstatement varied as a function of dose such that priming with the conditioning dose did not reinstate CPP, but reinstatement was observed following priming doses of METH that were either lower or higher than the conditioning dose. Prior NIC exposure had no effect on METH CPP, extinction, or reinstatement. Interestingly, at a METH dose (0.5 mg/kg) that did not induce reinstatement alone, acute NIC (0.2 mg/kg) in combination with METH induced reinstatement, suggesting that NIC produced a leftward shift in the dose-response effect of METH to reinstate CPP. These studies indicate that prior NIC exposure may not be necessary for enhancement of the rewarding effects of METH, in contrast to previous self-administration reports.

Sex differences in caffeine neurotoxicity following chronic ethanol exposure and withdrawal.

AIMS: Caffeine is a central nervous system stimulant that produces its primary effects via antagonism of the A(1) and A(2A) adenosine receptor subtypes. Previous work demonstrated a sex difference in neurotoxicity produced by specific adenosine A(1) receptor antagonism during ethanol withdrawal (EWD) in vitro that was attributable to effects downstream of A(1) receptors at NMDA receptors. The current studies were designed to examine the effect of non-specific adenosine receptor antagonism with caffeine during ethanol withdrawal on hippocampal toxicity in cultures derived from male and female rats. METHODS: At 5 days in vitro (DIV), half of the male and female organotypic hippocampal slice cultures were exposed to 50 mM ethanol (EtOH) in culture media for 10 days before exposure to caffeine (5, 20 and 100 microM) for the duration of a 24 h EWD period. In keeping with this timeline, the remaining ethanol-naïve cultures were given media changes at 10 and 15 DIV and exposed to caffeine (5, 20 and 100 microM) for 24 h at 15 DIV. Cytotoxicity was assessed by fluorescent microscopy and quantification of propidium iodide (PI) uptake in the pyramidal cell layers of the CA1 and CA3 regions and the granule cell layer of the dentate gyrus (DG). A two-way (sex x treatment) ANOVA was conducted within each hippocampal region. RESULTS: Twenty-four-hour withdrawal from 10-day exposure to 50 mM ethanol did not produce increased PI uptake in any hippocampal region. Caffeine exposure (5, 20 and 100 microM) in ethanol-naïve cultures did not produce toxicity in the DG or CA1 region, but 20 microM caffeine produced modest toxicity in the CA3 region. Exposure to 20 microM caffeine during EWD produced cytotoxicity in all hippocampal regions, though toxicity was sex-dependent in the DG and CA1 region. In the DG, both 5 and 20 microM caffeine produced significantly greater PI uptake in ethanol-exposed female cultures compared to ethanol-naïve female cultures and all male cultures. Similarly, 20 microM caffeine caused markedly greater toxicity in female cultures as compared to male cultures in the CA1 region. CONCLUSIONS: Twenty-four-hour exposure to caffeine during EWD produced significant toxicity in the pyramidal cell layer of the CA3 region in male and female cultures, though toxicity in the granule cell layer of the DG and pyramidal cell layer of the CA1 region was observed only in female cultures. Greater sensitivity of the female slice cultures to toxicity upon caffeine exposure after prolonged ethanol exposure is consistent with previous studies of effects of a specific A(1) receptor antagonism during EWD on toxicity and indicate that this effect is independent of the hormonal milieu. Together, these data suggest that the A(1) receptor subtype is predominant in mediating caffeine's neurotoxic effects during EWD. These findings demonstrate the importance of considering gender/sex when examining neuroadaptive changes in response to ethanol exposure and withdrawal.