Effects of nicotine on alcohol drinking in female mice selectively-bred for high or low alcohol preference

Background Studies show that repeated nicotine use associates with high alcohol consumption in humans, and that nicotine exposure sometimes increases alcohol consumption in animal models. However, the relative roles of genetic predisposition to high alcohol consumption, the alcohol drinking patterns, and the timing of nicotine exposure both with respect to alcohol drinking and developmental stage remain unclear. The studies here manipulated all these variables, using mice selectively bred for differences in free-choice alcohol consumption to elucidate the role of genetics and nicotine exposure in alcohol consumption behaviors. Methods In Experiments 1 and 2, we assessed the effects of repeated nicotine (0, 0.5 or 1.5 mg/kg) injections immediately before binge-like (drinking-in-the-dark; Experiment 1) or during free-choice alcohol access (Experiment 2) on these alcohol drinking behaviors (immediately after injections and during re-exposure to alcohol access 14 days later) in adult high- (HAP2) and low-alcohol preferring (LAP2) female mice (co-exposure model). In Experiments 3 and 4, we assessed the effects of repeated nicotine (0, 0.5 or 1.5 mg/kg) injections 14 days prior to binge-like and free-choice alcohol access on these alcohol drinking behaviors in adolescent HAP2 and LAP2 female mice (Experiment 3) or adult HAP2 female mice (Experiment 4). Results In Experiment 1, we found that repeated nicotine (0.5 and 1.5 mg/kg) and alcohol co-exposure significantly increased binge-like drinking behavior in HAP2 but not LAP2 mice during the re-exposure phase after a 14-day abstinence period. In Experiment 2, 1.5 mg/kg nicotine injections significantly reduced free-choice alcohol intake and preference in the 3rd hour post-injection in HAP2 but not LAP2 mice. No significant effects of nicotine treatment on binge-like or free-choice alcohol drinking were observed in Experiments 3 and 4. Conclusions These results show that the temporal parameters of nicotine and alcohol exposure, pattern of alcohol access, and genetic predisposition for alcohol preference influence nicotine's effects on alcohol consumption. These findings in selectively bred mice suggest that humans with a genetic history of alcohol-use disorders may be more vulnerable to develop nicotine and alcohol co-use disorders

Generation of a CRF1-Cre transgenic rat and the role of central amygdala CRF1 cells in nociception and anxiety-like behavior

Corticotropin-releasing factor type-1 (CRF1) receptors are critical to stress responses because they allow neurons to respond to CRF released in response to stress. Our understanding of the precise role of CRF1-expressing neuronal populations in CRF-mediated behaviors has been largely limited to mouse experiments due to the lack of genetic tools available to selectively visualize and manipulate CRF1+ cells in rats. Here, we describe the generation and validation of a transgenic CRF1-Cre-tdTomato rat, which expresses a bicistronic iCre-2A-tdTomato transgene directed by 200kb of promoter and enhancer sequence surrounding the Crhr1 cDNA present within a BAC clone, that has been transgenically inserted into the rat genome. We report that Crhr1 and Cre mRNA expression are highly colocalized in CRF1-Cre-tdTomato rats within both the central amygdala (CeA), composed of mostly GABAergic neurons, and in the basolateral amygdala (BLA), composed of mostly glutamatergic neurons. In the CeA, membrane properties, inhibitory synaptic transmission, and responses to CRF bath application in tdTomato+ neurons are similar to those previously reported in GFP+ cells in CRFR1-GFP mice. We show that stimulatory DREADD receptors can be selectively targeted to CeA CRF1+ cells via virally delivered Cre-dependent transgenes, that transfected Cre/tdTomato+ cells are activated by clozapine-n-oxide in vitro and in vivo, and that activation of these cells in vivo increases anxiety-like behavior and nocifensive responses. Outside the amygdala, we show that Cre-tdTomato is expressed in several brain areas across the rostrocaudal axis of the CRF1-Cre-tdTomato rat brain, and that the expression pattern of Cre-tdTomato cells is similar to the known expression pattern of CRF1 cells. Given the accuracy of expression in the CRF1-Cre rat, modern genetic techniques used to investigate the anatomy, physiology, and behavioral function of CRF1+ neurons and circuits can now be performed in assays that require the use of rats as the model organism

World Health Organization cardiovascular disease risk charts: revised models to estimate risk in 21 global regions

BACKGROUND: To help adapt cardiovascular disease risk prediction approaches to low-income and middle-income countries, WHO has convened an effort to develop, evaluate, and illustrate revised risk models. Here, we report the derivation, validation, and illustration of the revised WHO cardiovascular disease risk prediction charts that have been adapted to the circumstances of 21 global regions. METHODS: In this model revision initiative, we derived 10-year risk prediction models for fatal and non-fatal cardiovascular disease (ie, myocardial infarction and stroke) using individual participant data from the Emerging Risk Factors Collaboration. Models included information on age, smoking status, systolic blood pressure, history of diabetes, and total cholesterol. For derivation, we included participants aged 40-80 years without a known baseline history of cardiovascular disease, who were followed up until the first myocardial infarction, fatal coronary heart disease, or stroke event. We recalibrated models using age-specific and sex-specific incidences and risk factor values available from 21 global regions. For external validation, we analysed individual participant data from studies distinct from those used in model derivation. We illustrated models by analysing data on a further 123 743 individuals from surveys in 79 countries collected with the WHO STEPwise Approach to Surveillance. FINDINGS: Our risk model derivation involved 376 177 individuals from 85 cohorts, and 19 333 incident cardiovascular events recorded during 10 years of follow-up. The derived risk prediction models discriminated well in external validation cohorts (19 cohorts, 1 096 061 individuals, 25 950 cardiovascular disease events), with Harrell's C indices ranging from 0·685 (95% CI 0·629-0·741) to 0·833 (0·783-0·882). For a given risk factor profile, we found substantial variation across global regions in the estimated 10-year predicted risk. For example, estimated cardiovascular disease risk for a 60-year-old male smoker without diabetes and with systolic blood pressure of 140 mm Hg and total cholesterol of 5 mmol/L ranged from 11% in Andean Latin America to 30% in central Asia. When applied to data from 79 countries (mostly low-income and middle-income countries), the proportion of individuals aged 40-64 years estimated to be at greater than 20% risk ranged from less than 1% in Uganda to more than 16% in Egypt. INTERPRETATION: We have derived, calibrated, and validated new WHO risk prediction models to estimate cardiovascular disease risk in 21 Global Burden of Disease regions. The widespread use of these models could enhance the accuracy, practicability, and sustainability of efforts to reduce the burden of cardiovascular disease worldwide. FUNDING: World Health Organization, British Heart Foundation (BHF), BHF Cambridge Centre for Research Excellence, UK Medical Research Council, and National Institute for Health Research

Nicotine Consumption and Motivation-Related Responses in High and Low Alcohol Preferring Mice

Alcohol and tobacco use behaviors are highly correlated within individuals and studies have shown that these behaviors are genetically influenced. Also, initial sensitivity to the rewarding and aversive effects of these drugs are strong predictors of subsequent use. The purpose of this study is to assess the genetic relationship between alcohol and nicotine consumption, reward, and aversion using mice selectively-bred for high (HAP) or low alcohol preference (LAP). HAP mice have been previously shown to less sensitive to both the rewarding and aversive effects of alcohol, as measured by conditioned place preference (CPP) and conditioned taste aversion (CTA), compared to LAP mice. Here, free-choice nicotine oral consumption, nicotine-induced CPP, and nicotine-induced CTA were assessed in HAP and LAP mice. In addition, nicotine-induced changes in nucleus accumbens and striatum levels of dopamine, serotonin, and norepinephrine were assessed as potential neural correlates of any behavioral differences between HAP and LAP mice. HAP mice showed greater nicotine intake and intake ratio, and lower sensitivity to nicotine-induced CTA, compared to LAP mice. There were no line differences in nicotine-induced CPP or in any of the neurochemicals measured. These results suggest that the genetic factors that permit higher alcohol and nicotine consumption also confer lower sensitivity to the aversive effects of these drugs

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

<div><p>Nicotinic acetylcholine receptors containing α4 subunits (α4β2* 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.</p></div

Functional deletion of α4 subunits in VTA.

<p>a) Recordings were made from α4-flox;Cre(-) and Cre(+) VTA DA neurons. ACh (1 mM) was applied by pressure ejection and inward currents were recorded. All recorded traces are shown in gray, and an averaged trace is shown in blue. Scale bar: 40 pA, 400 ms. b) Quantification of ACh-evoked currents in AAV-infected mice. Mean inward ACh-evoked currents from α4-flox;Cre(-) (n = 19 cells from n = 9 animals), α4-flox;Cre(+) (n = 14 cells from n = 5 animals), WT;Cre(-) (n = 7 cells from n = 2 animals), and WT;Cre(+) (n = 8 cells from n = 2 animals) cells ± SEM. ****<i>p</i><0.0001 (unpaired <i>t</i>-test, <i>t</i> = 8.788). c) Residual ACh-evoked currents in α4-flox;Cre(+) neurons are αCtxMII-sensitive. For Cre(+) responses in (B), αCtxMII (100 nM) was applied by superfusion and ACh was re-applied after 12–15 min. Before-after responses for n = 4 αCtxMII-treated neurons (n = 2 animals) are shown. **<i>p</i> = 0.0052 (paired <i>t</i>-test, <i>t</i> = 7.348).</p

Nicotine CPP is reduced in mice with vMB α4 deletion.

<p>a) CPP schematic. Prior to a 5-day, biased CPP procedure to establish nicotine CPP, mice were mildly food-restricted and handled. Drug-free pre-test and post-test days flanked 3 consecutive conditioning days that consisted of morning and afternoon saline (SAL) and nicotine (NIC) conditioning sessions. b) Nicotine CPP in C57Bl/6 WT mice. Groups of WT mice were conditioned with saline (n = 12), 0.25 mg/kg NIC (n = 12), or 0.5 mg/kg NIC (n = 12) to validate our CPP assay and identify a dose to be used subsequently in α4-flox mice. Mean (± SEM) place preference score is shown for the three drug doses. <i>p</i> values are for Dunnett’s multiple comparisons test. c) Nicotine CPP in α4-flox mice. AAV-GFP or AAV-Cre-GFP vectors were infused into vMB of α4-flox mice (GFP(+), n = 10; Cre(+), n = 10), and mice were subsequently conditioned with 0.25 mg/kg NIC. Mean (± SEM) place preference score is shown for both groups. <i>p</i> value is for unpaired t-test. d) Mean (± SEM) locomotor activity during the pre-test and post-test is shown for α4-flox mice conditioned with NIC. e) Mean (± SEM) locomotor activity during conditioning sessions 1, 2, and 3 is shown for α4-flox mice conditioned with NIC.</p

Cre virus infection of VTA DA and GABA neurons.

<p>a) Targeting the VTA with AAV vectors. Mice were infused into the VTA with AAV-Cre-GFP vectors. After 2–3 weeks, mice were perfused and brains were sectioned for immunohistochemistry. Anti-GFP staining identifies infected neurons, while anti-TH staining identifies DA neurons. High magnification (60X) images show Cre-GFP expression in TH(+) and TH(-) neurons in VTA, indicated by one or two arrows, respectively. IPN: interpeduncular nucleus, ML: medial lemniscus. b) Cre recombinase expression in VTA GABA neurons. Slices from the infection shown in (a) were stained with anti-GAD65/67 antibodies. Native GFP signal was imaged without anti-GFP staining.</p

Conditional KO of α4 nAChR subunits.

<p>a) α4-flox mice have loxP sites flanking exon 5 of the α4 nAChR gene. Infusion of viral vectors expressing Cre recombinase into the VTA results in conditional deletion of α4 in infected cells. b) The three main heteromeric nAChR subtypes found in neurons of the midbrain DA system, α4α6β2*, α6 β2(non-α4)*, and α4β2(non-α6)*, are shown. Deletion of α4 eliminates α4α6β2* and α4β2(non-α6)* subtypes, while isolating the activity of α6β2(non-α4)* nAChRs. c) Viral spread of AAV-GFP vectors. An AAV-GFP virus was infused bilaterally into the VTA of a C57BL/6 WT mouse, followed 3 weeks later by perfusion and anti-GFP staining of brain sections.</p