Aversion-associated drug and alcohol seeking in females.

Compulsive drug intake is characterized by the continuation of use regardless of negative consequences. This is modeled preclinically using procedures where a negative stimulus is delivered contingently with consumption of the reinforcer. In humans, women and men exhibit different drug taking behavior as it pertains to overall use, withdrawal symptoms, and rate of dependence. In substance use research, females have often been excluded from many studies due to concerns that circulating sex hormones may affect drug seeking behavior. However, the more recent inclusion of females in preclinical studies has identified interesting sex differences in aversion-resistant intake of drugs and alcohol. This review will serve to summarize key findings in aversion-related intake of alcohol, psychostimulants, and opioids in females by examining studies that have included female subjects. Further discussion will examine the effect of intake model, neuroanatomical pathways, and sex hormones in the expression of aversion-resistant drug and alcohol consumption.

Sex differences in neuronal activation in the cortex and midbrain during quinine-adulterated alcohol intake.

AIMS: Continued alcohol consumption despite negative consequences is a core symptom of alcohol use disorder. This is modeled in mice by pairing negative stimuli with alcohol, such as adulterating alcohol solution with quinine. Mice consuming alcohol under these conditions are considered to be engaging in aversion-resistant intake. Previously, we have observed sex differences in this behavior, with females more readily expressing aversion-resistant consumption. We also identified three brain regions that exhibited sex differences in neuronal activation during quinine-alcohol drinking: ventromedial prefrontal cortex (vmPFC), posterior insular cortex (PIC), and ventral tegmental area (VTA). Specifically, male mice showed increased activation in vmPFC and PIC, while females exhibited increased activation in VTA. In this study, we aimed to identify what specific type of neurons are activated in these regions during quinine-alcohol drinking. METHOD: We assessed quinine-adulterated alcohol intake using the two-bottle choice procedure. We also utilized RNAscope in situ hybridization in the three brain regions that previously exhibited a sex difference to examine colocalization of Fos, glutamate, GABA, and dopamine. RESULT: Females showed increased aversion-resistant alcohol consumption compared to males. We also found that males had higher colocalization of glutamate and Fos in vmPFC and PIC, while females had greater dopamine and Fos colocalization in the VTA. CONCLUSIONS: Collectively, these experiments suggest that glutamatergic output from the vmPFC and PIC may have a role in suppressing, and dopaminergic activity in the VTA may promote, aversion-resistant alcohol consumption. Future experiments will examine neuronal circuits that contribute to sex differences in aversion resistant consumption.

Vicarious defeat stress induces increased alcohol consumption in female mice: Role of neurokinin-1 receptor and interleukin-6.

There is a high frequency of comorbidity of alcohol use disorder (AUD) and depression in human populations. We have studied this relationship in our lab using the social defeat stress (SDS) model, which results in both depression-like behaviours and increased alcohol consumption in male mice. However, standard SDS procedures are difficult to use in female mice due to a lack of territorial aggression. In the experiments presented here, we used vicarious defeat stress (VDS) to assess social withdrawal and alcohol consumption in female C57BL6/J mice. We also assessed the expression of interleukin-6 (IL6), which is a proinflammatory cytokine that is associated with depression in humans and sensitivity to SDS in mice. In these experiments, C57BL/6 female mice underwent 10 days of VDS where they witnessed the physical defeat of a male conspecific by an aggressive CD1 mouse. After the end of VDS, mice were either given access to alcohol or sacrificed for the measurement of IL6 expression. We found that VDS increased alcohol consumption and IL6 expression in the frontal cortex and hippocampus. Given that the neurokinin-1 receptor (NK1R) can mediate both stress-induced alcohol consumption and IL6 expression, we tested the ability of NK1R antagonism to reduce VDS-induced alcohol consumption and found that this treatment reduced alcohol intake in both VDS-exposed mice and in unstressed controls. The observed increase in alcohol consumption suggests that VDS is a model that can be utilized to study stress-induced alcohol consumption in female mice, and that this is sensitive to NK1R antagonism.

Sex differences in neuronal activation during aversion-resistant alcohol consumption.

BACKGROUND: One of the DSM-5 criteria for Alcohol Use Disorder is continued alcohol consumption despite negative consequences. This has been modeled in mice using adulteration of alcohol solution with the bitter tastant quinine. Mice that continue to consume alcohol despite this adulteration are considered aversion resistant. The limited number of studies dissecting the underlying neuronal mechanisms of aversion-resistant drinking behaviors used only male subjects. We have previously shown that female mice are more resistant to quinine adulteration of alcohol than males. Our aim here is to identify potential sex differences in neuronal activation that may underlie this behavior. METHODS: Male and female C57BL/6J mice were allowed continuous access to 20% alcohol in a two-bottle choice procedure. To test aversion-resistance, the alcohol was adulterated with increasing concentrations (0.03, 0.1, and 0.2 mM) of quinine hydrochloride. After consumption rates were calculated, brains were extracted to examine neuronal activation using Fos immunohistochemistry. RESULTS: We found that female mice suppressed their intake to a lesser extent than males when the alcohol solution was adulterated with quinine. Our Fos staining revealed three regions of interest that exhibit a sex difference during quinine-adulterated alcohol drinking: the ventromedial prefrontal cortex (vmPFC), the posterior insular cortex (PIC), and the ventral tegmental area (VTA). Both the vmPFC and the PIC exhibited higher neuronal activation in males during quinine-adulterated alcohol consumption. However, females showed higher Fos activation in the VTA during quinine-adulterated alcohol consumption. CONCLUSIONS: Females more readily exhibit aversion-resistant alcohol intake than their male counterparts and exhibit some differences in neuronal activation patterns. We conclude that there are sex differences in neurocircuitry that may underlie compulsive drinking behaviors.

Estradiol mediates sex differences in aversion-resistant alcohol intake.

Introduction: Alcohol consumption despite negative consequences is a core symptom of alcohol use disorder. This can be modeled in mice by pairing aversive stimuli with alcohol consumption, such as adding the bitter tastant quinine to the alcohol solution. If an animal continues to drink alcohol despite such negative stimuli, this is typically considered aversion-resistant, or inflexible, drinking behavior. Previous studies in our lab have found that females are more aversion-resistant than males in that they tolerate higher concentrations of quinine before they suppress their alcohol intake. Interestingly, we did not observe any differences in intake across the estrous cycle. In regards to neuronal activation patterns during quinine-alcohol intake, we have found that male mice show higher levels of activation in the ventromedial prefrontal cortex and posterior insular cortex, while females show higher levels of activation in the ventral tegmental area. Methods: In the experiments presented here, we conducted ovariectomies to further examine the role of circulating sex hormones in aversion-resistant alcohol intake and neuronal activation patterns. Furthermore, we used hormonal addback of estradiol or progesterone to determine which ovarian sex hormone mediates aversion-resistant consumption. Results: We found that ovariectomy reduced quinine-adulterated alcohol intake, demonstrating that circulating sex hormones play a role in this behavior. We also observed reduced neuronal activation in the VTA of ovariectomized mice compared to sham females, and that estradiol supplementation reversed the effect of ovariectomy on quinine-alcohol intake. Discussion: Taken together with our prior data, these findings suggest that circulating estradiol contributes to the expression of aversion-resistant alcohol intake and neuronal activity in the VTA. However, since this behavior is not affected by the estrous cycle, we believe this is due to a threshold level of this hormone, as opposed to fluctuations that occur across the estrous cycle.

The effect of concurrent access to alcohol and oxycodone on self-administration and reinstatement in rats.

RATIONALE: Although polysubstance use is highly prevalent, preclinical studies that assess voluntary consumption of multiple substances at the same time are rather uncommon. Overlooking drug taking patterns commonly observed in humans may limit the translational value of preclinical models. OBJECTIVES: Here, we aimed to develop a model of polysubstance use that could be used to assess oral operant self-administration patterns under concurrent access to alcohol and the prescription opioid oxycodone. METHODS: After a training period where animals associated specific cues and levers with each drug, rats self-administered alcohol and oxycodone solutions concurrently in daily sessions. Oxycodone was then removed to assess potential changes in alcohol consumption. The role of cues and stress on alcohol consumption and oxycodone seeking was also examined under reinstatement conditions. RESULTS: We found that females consumed more alcohol and oxycodone than males when given access to both drugs, and this effect on alcohol intake persisted when oxycodone was removed. Additionally, re-exposure to oxycodone cues in combination with the administration of the pharmacological stressor yohimbine drove reinstatement of oxycodone seeking in females but did not have a strong effect in males, possibly due to low levels of oxycodone intake during active self-administration in male rats. Additionally, yohimbine drove increased alcohol consumption, in line with prior findings from our group and others. CONCLUSIONS: Taken together, this study demonstrates that rats will concurrently self-administer both oxycodone and alcohol in operant chambers, and this procedure can serve as a platform for future investigations in polysubstance use and relapse-like behavior.

Which cyclin E prevails as prognostic marker for breast cancer? Results from a retrospective study involving 635 lymph node-negative breast cancer patients.

PURPOSE: To evaluate the prognostic value of cyclin E with a quantitative method for lymph node-negative primary breast cancer patients. PATIENTS AND METHODS: mRNA transcripts of full-length and splice variants of cyclin E1 (CCNE1) and cyclin E2 (CCNE2) were measured by real-time PCR in frozen tumor samples from 635 lymph node-negative breast cancer patients who had not received neoadjuvant or adjuvant systemic therapy. RESULTS: None of the PCR assays designed for the specific splice variants of the cyclins gave additional prognosis-related information compared with the common assays able to detect all variants. In Cox multivariate analysis, corrected for the traditional prognostic factors, high levels of cyclin E were independently associated with a short distant metastasis-free survival [hazard ratio (HR), 3.40; P < 0.001 for CCNE1 and HR, 1.76; P < 0.001 for CCNE2, respectively]. After dichotomizing the tumors at the median level of 70% tumor cells, the multivariate analysis showed particularly strong results for CCNE1 in the group of 433 patients with stroma-enriched primary tumors (HR, 5.12; P < 0.001). In these tumors, the worst prognosis was found for patients with estrogen receptor-negative tumors expressing high CCNE1 (HR, 9.89; P < 0.001) and for patients with small (T1) tumors expressing high CCNE1 (HR, 8.47; P < 0.001). CONCLUSION: Our study shows that both CCNE1 and CCNE2 qualify as independent prognostic markers for lymph node-negative breast cancer patients, and that CCNE1 may provide additional information for specific subgroups of patients.

Dysbindin Deficiency Modifies the Expression of GABA Neuron and Ion Permeation Transcripts in the Developing Hippocampus.

The neurodevelopmental factor dysbindin is required for synapse function and GABA interneuron development. Dysbindin protein levels are reduced in the hippocampus of schizophrenia patients. Mouse dysbindin genetic defects and other mouse models of neurodevelopmental disorders share defective GABAergic neurotransmission and, in several instances, a loss of parvalbumin-positive interneuron phenotypes. This suggests that mechanisms downstream of dysbindin deficiency, such as those affecting GABA interneurons, could inform pathways contributing to or ameliorating diverse neurodevelopmental disorders. Here we define the transcriptome of developing wild type and dysbindin null Bloc1s8sdy/sdy mouse hippocampus in order to identify mechanisms downstream dysbindin defects. The dysbindin mutant transcriptome revealed previously reported GABA parvalbumin interneuron defects. However, the Bloc1s8sdy/sdy transcriptome additionally uncovered changes in the expression of molecules controlling cellular excitability such as the cation-chloride cotransporters NKCC1, KCC2, and NCKX2 as well as the potassium channel subunits Kcne2 and Kcnj13. Our results suggest that dysbindin deficiency phenotypes, such as GABAergic defects, are modulated by the expression of molecules controlling the magnitude and cadence of neuronal excitability.

How ADAM-9 and ADAM-11 differentially from estrogen receptor predict response to tamoxifen treatment in patients with recurrent breast cancer: a retrospective study.

PURPOSE: To evaluate the predictive value of the disintegrin and metalloproteinases, ADAM-9, ADAM-10, ADAM-11, and ADAM-12, and of the matrix metalloproteinases, MMP-2 and MMP-9, in patients with recurrent breast cancer treated with tamoxifen. EXPERIMENTAL DESIGN: A retrospective study was done on 259 frozen specimens of estrogen receptor-positive primary breast carcinomas from patients who developed recurrent disease and were treated with tamoxifen as the first line of therapy. The expression levels of the biological factors were assessed by real-time quantitative reverse transcriptase PCR. RESULTS: Using log-transformed continuous variables, increasing levels of ADAM-9 [odds ratio (OR) = 1.41; P = 0.015] and decreasing levels of MMP-9 (OR, 0.81; P = 0.035) predicted favorable disease control independent from the traditional predictive factors. Furthermore, when tumors were dichotomized at the median level of 70% tumor cell nuclei, our univariate analysis showed particularly strong results for the group of 153 patients with primary tumors containing 30% or more stromal cells. Although estrogen receptor levels lost their predictive power for this group of patients, high levels of ADAM-9 (OR, 1.59; P = 0.007) and ADAM-11 (OR, 1.65; P = 0.001) were significantly associated with a higher efficacy of tamoxifen therapy. CONCLUSIONS: Our results show that especially for primary tumors containing stromal elements, the assessment of mRNA expression levels of ADAM-9 and ADAM-11 could be useful to identify patients with recurrent breast cancer who are likely to benefit or fail from tamoxifen therapy.

The Endosome Localized Arf-GAP AGAP1 Modulates Dendritic Spine Morphology Downstream of the Neurodevelopmental Disorder Factor Dysbindin.

AGAP1 is an Arf1 GTPase activating protein that interacts with the vesicle-associated protein complexes adaptor protein 3 (AP-3) and Biogenesis of Lysosome Related Organelles Complex-1 (BLOC-1). Overexpression of AGAP1 in non-neuronal cells results in an accumulation of endosomal cargoes, which suggests a role in endosome-dependent traffic. In addition, AGAP1 is a candidate susceptibility gene for two neurodevelopmental disorders, autism spectrum disorder (ASD) and schizophrenia (SZ); yet its localization and function in neurons have not been described. Here, we describe that AGAP1 localizes to axons, dendrites, dendritic spines and synapses, colocalizing preferentially with markers of early and recycling endosomes. Functional studies reveal overexpression and down-regulation of AGAP1 affects both neuronal endosomal trafficking and dendritic spine morphology, supporting a role for AGAP1 in the recycling endosomal trafficking involved in their morphogenesis. Finally, we determined the sensitivity of AGAP1 expression to mutations in the DTNBP1 gene, which is associated with neurodevelopmental disorder, and found that AGAP1 mRNA and protein levels are selectively reduced in the null allele of the mouse ortholog of DTNBP1. We postulate that endosomal trafficking contributes to the pathogenesis of neurodevelopmental disorders affecting dendritic spine morphology, and thus excitatory synapse structure and function.

Safety and immunogenicity of a recombinant Plasmodium falciparum AMA1 malaria vaccine adjuvanted with Alhydrogel, Montanide ISA 720 or AS02.

BACKGROUND: Plasmodium falciparum Apical Membrane Antigen 1 (PfAMA1) is a candidate vaccine antigen expressed by merozoites and sporozoites. It plays a key role in red blood cell and hepatocyte invasion that can be blocked by antibodies. METHODOLOGY/PRINCIPAL FINDINGS: We assessed the safety and immunogenicity of recombinant PfAMA1 in a dose-escalating, phase Ia trial. PfAMA1 FVO strain, produced in Pichia pastoris, was reconstituted at 10 microg and 50 microg doses with three different adjuvants, Alhydrogel, Montanide ISA720 and AS02 Adjuvant System. Six randomised groups of healthy male volunteers, 8-10 volunteers each, were scheduled to receive three immunisations at 4-week intervals. Safety and immunogenicity data were collected over one year. Transient pain was the predominant injection site reaction (80-100%). Induration occurred in the Montanide 50 microg group, resulting in a sterile abscess in two volunteers. Systemic adverse events occurred mainly in the AS02 groups lasting for 1-2 days. Erythema was observed in 22% of Montanide and 59% of AS02 group volunteers. After the second dose, six volunteers in the AS02 group and one in the Montanide group who reported grade 3 erythema (>50 mm) were withdrawn as they met the stopping criteria. All adverse events resolved. There were no vaccine-related serious adverse events. Humoral responses were highest in the AS02 groups. Antibodies showed activity in an in vitro growth inhibition assay up to 80%. Upon stimulation with the vaccine, peripheral mononuclear cells from all groups proliferated and secreted IFNgamma and IL-5 cytokines. CONCLUSIONS/SIGNIFICANCE: All formulations showed distinct reactogenicity profiles. All formulations with PfAMA1 were immunogenic and induced functional antibodies. TRIAL REGISTRATION: (Clinicaltrials.gov) NCT00730782.